Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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PYRANONES USEFUL AS ATM INHIBITORS
The present invention relates to compounds which act as ATM
inhibitors, their use and synthesis.
Human DNA is constantly under attack from reactive oxygen
intermediates principally from by-products of oxidative
metabolism. Reactive oxygen species are capable of producing
DNA single-strand breaks and, where two of these are generated
in close proximity, DNA double strand breaks (DSBs). In
addition, single- and double-strand breaks can be induced when
a DNA replication fork encounters a damaged template, and are
generated by exogenous agents such as ionising radiation (IR)
and certain anti-cancer drugs (e. g. bleomycin, etoposide,
camptothecin). DSBs also occur as intermediates in site-
specific V(D)J recombination, a process that is critical for
the generation of a functional vertebrate immune system. If
DNA DSBs are left unrepaired or are repaired inaccurately,
mutations and/or chromosomal aberrations are induced, which in
turn may lead to cell death. To combat the serious threats
posed by DNA DSBs, eukaryotic cells have evolved several
mechanisms to mediate their repair. Critical to the process
of DNA repair is the slowing down of cellular proliferation to
allow time for the cell to repair the damage. A key protein
in the detection of DNA DSBs and in the signalling of this
information to the cell cycle machinery is the kinase ATM
(ataxia telangiectasia mutated) (Durocher and Jackson (2001)
DNA-PK, ATM and ATR as sensors of DNA damage: variations on a
theme? Curr Opin Cell Biol., 13:225-31, Abraham (2001) Cell
cycle checkpoint signaling through the ATM and ATR kinases.
Genes Dev., 15; 2177-96).
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The ATM protein is an 350 kDa polypeptide that is a member
of the phosphatidylinositol (PI) 3-kinase family of proteins
by virtue of a putative kinase domain in its carboxyl-terminal
region (Savitsky et al (1995) A single ataxia telangiectasia
gene with a product similar to PI-3 kinase. Science, 268:1749-
53). Classical PI 3-kinases, such as PI 3-kinase itself, are
involved in signal transduction and phosphorylate inositol
lipids that act as intracellular second messengers (reviewed
in Toker and Cantley (1997) Signalling through the lipid
products of phosphoinositide-3-OH kinase, Nature, 387: 673-6).
However, ATM bears most sequence similarity with a subset of
the PI 3-kinase family that comprises proteins which, like
ATM, are involved in cell cycle control and/or in the
detection and signalling of DNA damage (Keith and Schreiber
(1995) PIK-related kinases: DNA repair, recombination, and
cell cycle checkpoints, Science, 270; 50-1, Zakian (1995)
ATM-related genes: what do they tell us about functions of the
human gene? Cell, 82; 685-7). Notably there is no evidence
to date that any members of this subset of the PI 3-kinase
family are able to phosphorylate lipids. However, all members
of this family have been shown to possess serine/threonine
kinase activity. ATM phosphorylates key proteins involved in
a variety of cell-cycle checkpoint signalling pathways that
are initiated in response to DNA DSBs production (see below).
These downstream effector proteins include p53, Chk2,
NBS1/nibrin, BRCA1 and Rad 17 (Abraham, 2001)
ATM is the product of the gene mutated in ataxia-
telangiectasia (A-T) (Savitsky et al (1995)). A-T is a human
autosomal recessive disorder present at an incidence of around
1 in 100,000 in the population. A-T is characterised by a
number of debilitating symptoms, including progressive
cerebellar degeneration, occulocutaneous telangiectasia,
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growth retardation, immune deficiencies, cancer predisposition
and certain characteristics of premature ageing (Lavin and
Shiloh (1997), The genetic defect in ataxia-telangiectasia.
Annu. Rev. Immunol., 15:177-202; Shiloh (2001), ATM and ATR:
networking cellular responses to DNA damage, Curr. Opin.
Genet. Dev., 11:71-7). At the cellular level, A-T is
characterised by a high degree of chromosomal instability,
radio-resistant DNA synthesis, and hypersensitivity to
ionizing radiation (IR) and radiomimetic drugs. In addition,
A-T cells are defective in the radiation induced Gl-S, S, and
G2-M cell cycle checkpoints that are thought to arrest the
cell cycle in response to DNA damage in order to allow repair
of the genome prior to DNA replication or mitosis (Lavin and
Shiloh, 1997). This may in part reflect the fact that A-T
cells exhibit deficient or severely delayed induction of p53
in response to IR. Indeed, p53-mediated downstream events are
also defective in A-T cells following IR exposure. ATM
therefore acts upstream of p53 in an IR-induced DNA damage
signalling pathway. A-T cells have also been shown to
accumulate DNA double-strand breaks (dsbs) after ionizing
radiation, suggesting a defect in dsb repair.
It is clear that ATM is a key regulator of the cellular
response to DNA DSBs. Therefore the inhibition of this kinase
through small molecules will sensitise cells to both ionising
radiation and to chemotherapeutics that induce DNA DSBs either
directly or indirectly. ATM inhibitors may thus be used as
adjuncts in cancer radiotherapy and chemotherapy. To date the
only reported inhibitors of ATM (caffeine and wortmannin;
Sarkaria, et al., (1999) Inhibition of ATM and ATR kinase
activities by the radiosensitizing agent, caffeine. Cancer
Res., 59:4375-82; Banin, et al., (1998) Enhanced
phosphorylation of p53 by ATM in response to DNA damage.
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Science, 281:1674-1677) do cause radiosensitisation but it is
unclear whether this mechanism of action is mediated through
ATM inhibition as these small molecules are very non-specific
in action as kinase inhibitors.
ATM function in response to ionising radiation induced DNA
damage has been shown to be tissue specific. For example,
while fibroblasts derived from Atm null mice are
radiosensitive Atm null neurons are radioresistant through a
lack of IR induced apoptosis (Herzog et al., (1998)
Requirement for Atm in ionizing radiation-induced cell death
in the developing central nervous system. Science, 280: 1089-
91). Therefore, inhibitors of ATM have the potential to be
radio-protective in specific cellular contexts.
ATM inhibitors may also prove useful in the treatment of
retroviral mediated diseases. It has been demonstrated that
ATM function is required to allow stable retroviral DNA
transduction under certain conditions (Daniel et al. (2001)
Wortmannin potentiates integrase-mediated killing of
lymphocytes and reduces the efficiency of stable transduction
by retroviruses. Mol. Cell Biol., 21: 1164-72). Therefore ATM
inhibitors have the potential to block retroviral DNA
integration.
ATM is known to play a crucial role in controlling the length
of telomeric chromosomal ends (Metcalfe et al. (1996)
Accelerated telomere shortening in ataxia telangiectasia. Nat
Genet., 13 :350-3). Telomeric ends in most normal cell types
shorten at each cell division. Cells with excessively
shortened telomeres are unable to divide. Inhibitors of ATM
may therefore, have utility in preventing cancer progression
by limiting the growth potential of cancerous or pre-cancerous
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cells. Furthermore, ATM does not appear to be part of the
telomerase enzyme itself (Metcalfe et al. (1996)) Therefore
it is likely that ATM inhibitors will work synergistically
with anti-telomerase drugs.
5
Cells derived from A-T patients or from mice null for ATM grow
slower in culture than genetically matched ATM positive cells.
Therefore an ATM inhibitor may have growth inhibitory/anti-
proliferative properties in its own right. Therefore an ATM
inhibitor may be used as a cytostatic agent in the treatment
of cancer.
A-T patients display immuno-deficiencies, demonstrating that
ATM is required for generation of a fully functional immune
system. Inhibitors of ATM may, therefore, be used in
modulating the immune system.
In summary ATM inhibitors have the potential to sensitise
tumour cells to ionising radiation or DNA DSB inducing
chemotherapeutics, to modulate telomere length control
mechanisms, to block retroviral integration, modulate the
immune system and to protect certain cell types from DNA
damage induced apoptosis.
The present inventors have now discovered compounds which
exhibit inhibition of ATM.
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Accordingly, the first aspect of the invention provides a
compound of formula I:
R'
R3 P N~ 2
R CI)
Q
Y
and isomers, salts, solvates, chemically protected forms, and
prodrugs thereof, wherein:
one of P and Q is O, and the other of P and Q is CH, where
there is a double bond between whichever of Q and P is CH and
the carbon atom bearing the R3 group;
Y is either O or S;
R1 and RZ are independently hydrogen, an optionally substituted
C1-~ alkyl group, C3-20 heterocyclyl group, or CS_ZO aryl group,
or may together form, along with the nitrogen atom to which
they are attached, an optionally substituted heterocyclic ring
having from 4 to 8 ring atoms;
R3 is a phenyl or pyridyl group, attached by a first bridge
group selected from -S-, -S(=O)-, -S(=0)2-, -O-, -NRN- and
CR~1R~2- to an optionally substituted CS_ZO carboaryl group, in
which one aromatic ring atom may be replaced by a nitrogen
ring atom;
the phenyl or pyridyl group and optionally substituted C5-2o
carboaryl group being optionally further linked by a second
bridge group, which is bound adjacent the first bridge group
on both groups so as to form an optionally substituted CS-~ ring
fused to both the phenyl or pyridyl group and the C5-20
carboaryl group, the phenyl or pyridyl group being further
optionally substituted;
wherein RN is selected from hydrogen, an ester group, an
optionally substituted C1_7 alkyl group, an optionally
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substituted C3-zo heterocyclyl group and an optionally
substituted C5-zo aryl group;
and R~1 and R~z are independently selected from hydrogen, an
optionally substituted C1-~ alkyl group, an optionally
substituted C3-zo heterocyclyl group and an optionally
substituted CS_zo aryl group.
Therefore, when P is O and Q is CH, the compound is of formula
(Ia)
R'
R3 O N ~ Z
R (la)
Y
and when P is CH and Q is O, the compound is of formula (Ib):
R'
3 I
R / NwRz
(Ib)
O
Y
A second aspect of the invention provides a composition
comprising a compound of the first aspect and a
pharmaceutically acceptable carrier or diluent.
A third aspect of the invention provides the use of a compound
of the first aspect in a method of therapy.
A fourth aspect of the invention provides the use of a
compound of the first aspect in the preparation of a
medicament for inhibiting the activity of ATM.
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A fifth aspect of the invention provides for the use of a
compound as defined in the first aspect of the invention in
the preparation of a medicament for use as an adjunct in
cancer therapy or for potentiating tumour cells for treatment
with ionising radiation or chemotherapeutic agents.
A sixth aspect of the invention provides for the use of a
compound as defined in the first aspect of the invention in
the preparation of a medicament for the treatment of
retroviral mediated diseases or disease ameliorated by the
inhibition of ATM, which include acquired immunodeficiency
syndrome.
A further aspect of the invention provides an active compound
as described herein for use in a method of treatment of the
human or animal body, preferably in the form of a
pharmaceutical composition.
Another aspect of the invention provides a method of
inhibiting ATM in vitro or in vivo, comprising contacting a
cell with an effective amount of an active compound as
described herein.
Definitions
Cl_~ alkyl: The term "C1_~alkyl", as used herein, pertains to a
monovalent moiety obtained by removing a hydrogen atom from a
Cl_~ hydrocarbon compound having from 1 to 7 carbon atoms, which
may be aliphatic or alicyclic, or a combination thereof, and
which may be saturated, partially unsaturated, or fully
unsaturated.
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Examples of saturated linear C1_~ alkyl groups include, but are
not limited to, methyl, ethyl, n-propyl, n-butyl, and n-pentyl
( amyl ) .
Examples of saturated branched C1_~ alkyl groups include, but
are not limited to, iso-propyl, iso-butyl, sec-butyl,
tert-butyl, and neo-pentyl.
Examples of saturated alicyclic C1_~ alkyl groups (also referred
to as "C3_7 cycloalkyl" groups) include, but are not limited to,
groups such as cyclopropyl, cyclobutyl, cyclopentyl, and
cyclohexyl, as well as substituted groups (e. g., groups which
comprise such groups), such as methylcyclopropyl,
dimethylcyclopropyl, methylcyclobutyl, dimethylcyclobutyl,
methylcyclopentyl, dimethylcyclopentyl, methylcyclohexyl,
dimethylcyclohexyl, cyclopropylmethyl and cyclohexylmethyl.
Examples of unsaturated Cl_7 alkyl groups which have one or more
carbon-carbon double bonds (also referred to as "C2_~alkenyl"
groups) include, but are not limited to, ethenyl (vinyl, -
CH=CHZ), 2-propenyl (allyl, -CH-CH=CHZ), isopropenyl
(-C(CH3)=CHZ), butenyl, pentenyl, and hexenyl.
Examples of unsaturated C1_~ alkyl groups which have one or more
carbon-carbon triple bonds (also referred to as "CZ_~ alkynyl"
groups) include, but are not limited to, ethynyl (ethinyl) and
2-propynyl (propargyl).
Examples of unsaturated alicyclic (carbocyclic) C1_~ alkyl
groups which have one or more carbon-carbon double bonds (also
referred to as "C3_~ cycloalkenyl" groups) include, but are not
limited to, unsubstituted groups such as cyclopropenyl,
cyclobutenyl, cyclopentenyl, and cyclohexenyl, as well as
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substituted groups (e. g., groups which comprise such groups)
such as cyclopropenylmethyl and cyclohexenylmethyl.
Cs-zo heterocyclyl: The term "C3_zo heterocyclyl", as used herein,
5 pertains to a monovalent moiety obtained by removing a
hydrogen atom from a ring atom of a C3_zo heterocyclic compound,
said compound having one ring, or two or more rings (e. g.,
spiro, fused, bridged), and having from 3 to 20 ring atoms,
atoms, of which from 1 to 10 are ring heteroatoms, and wherein
10 at least one of said rings) is a heterocyclic ring.
Preferably, each ring has from 3 to 7 ring atoms, of which
from 1 to 4 are ring heteroatoms. ~~C3_zo" denotes ring atoms,
whether carbon atoms or heteroatoms.
Examples of C3_zo heterocyclyl groups having one nitrogen ring
atom include, but are not limited to, those derived from
aziridine, azetidine, pyrrolidines (tetrahydropyrrole),
pyrroline (e. g., 3-pyrroline, 2,5-dihydropyrrole), 2H-pyrrole
or 3H-pyrrole (isopyrrole, isoazole), piperidine,
dihydropyridine, tetrahydropyridine, and azepine.
Examples of C3_zo heterocyclyl groups having one oxygen ring
atom include, but are not limited to, those derived from
oxirane, oxetane, oxolane (tetrahydrofuran), oxole
(dihydrofuran), oxane (tetrahydropyran), dihydropyran, pyran
(C6), and oxepin. Examples of substituted C3_zo heterocyclyl
groups include sugars, in cyclic form, for example, furanoses
and pyranoses, including, for example, ribose, lyxose, xylose,
galactose, sucrose, fructose, and arabinose.
Examples of C3_zo heterocyclyl groups having one sulphur ring
atom include, but are not limited to, those derived from
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thiirane, thietane, thiolane (tetrahydrothiophene), thiane
(tetrahydrothiopyran), and thiepane.
Examples of C3_ZO heterocyclyl groups having two oxygen ring
atoms include, but are not limited to, those derived from
dioxolane, dioxane, and dioxepane.
Examples of C3_ZO heterocyclyl groups having two nitrogen ring
atoms include, but are not limited to, those derived from
imidazolidine, pyrazolidine (diazolidine), imidazoline,
pyrazoline (dihydropyrazole), and piperazine.
Examples of C3_zo heterocyclyl groups having one nitrogen ring
atom and one oxygen ring atom include, but are not limited to,
those derived from tetrahydrooxazole, dihydrooxazole,
tetrahydroisoxazole, dihydroisoxazole, morpholine,
tetrahydrooxazine, dihydrooxazine, and oxazine.
Examples of C3_ZO heterocyclyl groups having one oxygen ring
atom and one sulphur ring atom include, but are not limited
to, those derived from oxathiolane and oxathiane (thioxane).
Examples of C3_ZO heterocyclyl groups having one nitrogen ring
atom and one sulphur ring atom include, but are not limited
to, those derived from thiazoline, thiazolidine, and
thiomorpholine.
Other examples of C3_ZOheterocyclyl groups include, but are not
limited to, oxadiazine and oxathiazine.
Examples of heterocyclyl groups which additionally bear one or
more oxo (=O) groups, include, but are not limited to, those
derived from:
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C5 heterocyclics, such as furanone, pyrone, pyrrolidone
(pyrrolidinone), pyrazolone (pyrazolinone), imidazolidone,
thiazolone, and isothiazolone;
C6 heterocyclics, such as piperidinone (piperidone),
piperidinedione, piperazinone, piperazinedione, pyridazinone,
and pyrimidinone (e.g., cytosine, thymine, uracil), and
barbituric acid;
fused heterocyclics, such as oxindole, purinone (e. g.,
guanine), benzoxazolinone, benzopyrone (e. g., coumarin);
cyclic anhydrides (-C(=0)-0-C(=O)- in a ring), including
but not limited to malefic anhydride, succinic anhydride, and
glutaric anhydride;
cyclic carbonates (-0-C(=0)-0- in a ring), such as
ethylene carbonate and 1,2-propylene carbonate;
imides (-C(=O)-NR-C(=O)- in a ring), including but not
limited to, succinimide, maleimide, phthalimide, and
glutarimide;
lactones (cyclic esters, -0-C(=O)- in a ring), including,
but not limited to, (3-propiolactone, Y-butyrolactone,
b-valerolactone (2-piperidone), and ~-caprolactone;
lactams (cyclic amides, -NR-C(=0)- in a ring), including,
but not limited to, (3-propiolactam, y-butyrolactam
(2-pyrrolidone), b-valerolactam, and ~-caprolactam;
cyclic carbamates (-O-C(=0)-NR- in a ring), such as
2-oxazolidone;
cyclic ureas (-NR-C(=O)-NR- in a ring), such as
2-imidazolidone and pyrimidine-2,4-dione (e. g., thymine,
uracil).
C5-zo aryl: The term "CS_zo aryl", as used herein, pertains to a
monovalent moiety obtained by removing a hydrogen atom from an
aromatic ring atom of a C5_zo aromatic compound, said compound
having one ring, or two or more rings (e. g. fused), and having
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from 5 to 20 ring atoms, and wherein at least one of said
rings) is an aromatic ring. Preferably, each ring has from 5
to 7 ring atoms.
The ring atoms may be all carbon atoms, as in "carboaryl
groups", in which case the group may conveniently be referred
to as a "CS-zo carboaryl" group.
Examples of C5_zo aryl groups which do not have ring heteroatoms
(i.e. CS_zo carboaryl groups) include, but are not limited to,
those derived from benzene (i.e. phenyl) (C6), naphthalene
(Clo) , anthracene (C1q) , phenanthrene (C19) , naphthacene (C1$) ,
and pyrene (Cis) -
Examples of aryl groups which comprise fused rings, one of
which is not an aromatic ring, include, but are not limited
to, groups derived from indene and fluorene.
Alternatively, the ring atoms may include one or more
heteroatoms, including but not limited to oxygen, nitrogen,
and sulphur, as in "heteroaryl groups". In this case, the
group may conveniently be referred to as a "CS_zo heteroaryl"
group, wherein "C5_zo" denotes ring atoms, whether carbon atoms
or heteroatoms. Preferably, each ring has from 5 to 7 ring
atoms, of which from 0 to 9 are ring heteroatoms.
Examples of CS_zo heteroaryl groups include, but are not limited
to, C5 heteroaryl groups derived from furan (oxole), thiophene
(thiole), pyrrole (azole), imidazole (1,3-diazole), pyrazole
(1,2-diazole), triazole, oxazole, isoxazole, thiazole,
isothiazole, oxadiazole, and oxatriazole; and C6 heteroaryl
groups derived from isoxazine, pyridine (azine), pyridazine
(1,2-diazine), pyrimidine (1,3-diazine; e.g., cytosine,
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thymine, uracil), pyrazine (1,4-diazine), triazine, tetrazole,
and oxadiazole (furazan).
Examples of CS_ZO heteroaryl groups which comprise fused rings,
include, but are not limited to, C9 heterocyclic groups derived
from benzofuran, isobenzofuran, indole, isoindole, purine
(e.g., adenine, guanine), benzothiophene, benzimidazole; Cio
heterocyclic groups derived from quinoline, isoquinoline,
benzodiazine, pyridopyridine, quinoxaline; C13 heterocyclic
groups derived from carbazole, dibenzothiophene, dibenzofuran;
C1q heterocyclic groups derived from acridine, xanthene,
phenoxathiin, phenazine, phenoxazine, phenothiazine.
The above C1_~ alkyl, C3_ZO heterocyclyl, and CS_ZO aryl groups,
whether alone or part of another substituent, may themselves
optionally be substituted with one or more groups selected
from themselves and the additional substituents listed below.
Halo: -F, -Cl, -Br, and -I.
Hydroxy: -OH.
Ether: -OR, wherein R is an ether substituent, for example, a
C1_~ alkyl group (also referred to as a C1_~ alkoxy group,
discussed below), a C3_ZOheterocyclyl group (also referred to
as a C3_zo heterocyclyloxy group) , or a CS-2o aryl group (also
referred to as a CS_zo aryloxy group) , preferably a C1_7 alkyl
group.
C1-~ alkoxy: -OR, wherein R is a C1_~ alkyl group. Examples of
C1_~ alkoxy groups include, but are not limited to, -OCH3
(methoxy), -OCH2CH3 (ethoxy) and -OC(CH3)3 (tert-butoxy).
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C1_2 alkdioxylene: The term "C1_2 alkdioxylene, " as used herein,
pertains to a bidentate moiety obtained by removing two
hydrogen atoms from each of two different alcohol groups of a
C1_2 hydrocarbon diol compound having from 1 or 2 carbon atoms,
5 i . a . CHZ (OH ) z and HO-CHZ-CHZ-OH, to form -O-CH2-0- and -0-CHz-
CHZ-O-. This bidentate moiety may be the substituent group of
a single atom or of two adjacent atoms.
Oxo (keto, -one): =0. Examples of cyclic compounds and/or
10 groups having, as a substituent, an oxo group (=O) include,
but are not limited to, carbocyclics such as cyclopentanone
and cyclohexanone; heterocyclics, such as pyrone, pyrrolidone,
pyrazolone, pyrazolinone, piperidone, piperidinedione,
piperazinedione, and imidazolidone; cyclic anhydrides,
15 including but not limited to malefic anhydride and succinic
anhydride; cyclic carbonates, such as propylene carbonate;
imides, including but not limited to, succinimide and
maleimide; lactones (cyclic esters, -0-C(=0)- in a ring),
including, but not limited to, (3-propiolactone,
y-butyrolactone, ~-valerolactone, and ~-caprolactone; and
lactams (cyclic amides, -NH-C(=0)- in a ring), including, but
not limited to, (3-propiolactam, y-butyrolactam (2-
pyrrolidone), ~-valerolactam, and E-caprolactam.
Imino (imine): =NR, wherein R is an imino substituent, for
example, hydrogen, C1_~ alkyl group, a C3_ZOheterocyclyl group,
or a C5_zo aryl group, preferably hydrogen or a C1_~ alkyl group.
Examples of ester groups include, but are not limited to, =NH,
=NMe, =NEt, and =NPh.
Formyl (carbaldehyde, carboxaldehyde): -C(=O)H.
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Acyl (keto): -C(=O)R, wherein R is an acyl substituent, for
example, a C1_~alkyl group (also referred to as C1_~ alkylacyl or
C1_~ alkanoyl) , a C3_zo heterocyclyl group (also referred to as
C3_zo heterocyclylacyl) , or a CS_zo aryl group (also referred to
as CS_zo arylacyl) , preferably a C1_~ alkyl group. Examples of
aryl groups include, but are not limited to, -C(=O)CH3
(acetyl), -C(=0)CH2CH3 (propionyl), -C(=O)C(CH3)3 (butyryl), and
-C (=0) Ph (benzoyl, phenone) .
Carboxy (carboxylic acid): -COON.
Ester (carboxylate, carboxylic acid ester, oxycarbonyl):
-C(=0)OR, wherein R is an ester substituent, for example, a
C1_7 alkyl group, a C3_zo heterocyclyl group, or a C5_zo aryl
group, preferably a C1_~alkyl group. Examples of ester groups
include, but are not limited to, -C (=0) OCH3, -C (=O) OCHZCH3,
-C (=O) OC (CH3) 3, and -C (=O) OPh.
Acyloxy (reverse ester): -OC(=0)R, wherein R is an acyloxy
substituent, for example, a C1_~ alkyl group, a C3-zo
heterocyclyl group, or a C5_zo aryl group, preferably a C1_~alkyl
group. Examples of acyloxy groups include, but are not
limited to, -OC (=0) CH3 (acetoxy) , -OC (=0) CHZCH3, -OC (=0) C (CH3) 3,
-OC (=0) Ph, and -OC (=0) CH2Ph.
Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide):
-C (=0) NRlRz, wherein R1 and Rz are independently amino
substituents, as defined for amino groups. Examples of amido
groups include, but are not limited to, -C(=O)NHz, -C(=O)NHCH3,
-C (=0) N (CH3) z, -C (=O) NHCHZCH3, and -C (=0) N (CHZCH3) z, as well as
amido groups in which R1 and Rz, together with the nitrogen
atom to which they are attached, form a heterocyclic structure
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as in, for example, piperidinocarbonyl, morpholinocarbonyl,
thiomorpholinocarbonyl, and piperazinocarbonyl.
Acylamido (acylamino) : -NR1C (=0) Rz, wherein Rl is an amide
substituent, for example, hydrogen, a C1_~ alkyl group, a C3-zo
heterocyclyl group, or a CS_zo aryl group, preferably hydrogen
or a C1_~ alkyl group, and Rz is an acyl substituent, for
example, a C1_~ alkyl group, a C3-zo heterocyclyl group, or a C5_zo
aryl group, preferably hydrogen or a C1_~ alkyl group. Examples
of acylamide groups include, but are not limited to,
-NHC (=O) CH3 , -NHC (=0) CHZCH3, and -NHC (=O) Ph. R1 and Rz may
together form a cyclic structure, as in, for example,
succinimidyl, maleimidyl and phthalimidyl:
O N O O N O O N O
succinimidyl maleimidyl phthalimidyl
Thioamido ( thiocarbamyl ) : -C (=S ) NRlRz, wherein R1 and Rz are
independently amino substituents, as defined for amino groups.
Examples of amido groups include, but are not limited to,
-C (=S) NHz, -C (=S) NHCH3, -C (=S) N (CH3) z, and -C (=S) NHCHZCH3.
Tetrazolyl: a five membered aromatic ring having four nitrogen
atoms and one carbon atom,
~~N
N
N'
Amino: -NRIRz, wherein R1 and Rz are independently amino
substituents, for example, hydrogen, a C1_7 alkyl group (also
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18
referred to as C1_~ alkylamino or di-C1_~ alkylamino) , a C3_Zo
heterocyclyl group, or a C5_ZO aryl group, preferably H or a
C1_~alkyl group, or, in the case of a "cyclic" amino group, R1
and R2, taken together with the nitrogen atom to which they are
attached, form a heterocyclic ring having from 4 to 8 ring
atoms. Examples of amino groups include, but are not limited
to, -NH2, -NHCH3, -NHC (CH3) z, -N (CH3) 2, -N (CHZCH3) 2, and -NHPh.
Examples of cyclic amino groups include, but are not limited
to, aziridino, azetidino, pyrrolidino, piperidino, piperazino,
morpholino, and thiomorpholino.
Imino: =NR, wherein R is an imino substituent, for example,
hydrogen, a C1_~ alkyl group, a C3_ZO heterocyclyl group, or a
CS_ZO aryl group, preferably H or a C1_~ alkyl group.
Amidine: -C(=NR)NR2, wherein each R is an amidine substituent,
for example, hydrogen, a C1_~ alkyl group, a C3_ZO heterocyclyl
group, or a CS_ZO aryl group, preferably H or a C1_~ alkyl group.
An example of an amidine group is -C(=NH)NH2.
Nitro: -NOZ.
Nitroso: -N0.
Azido: -N3.
Cyano (nitrile, carbonitrile): -CN.
Isocyano: -NC.
Cyanato: -OCN.
Isocyanato: -NCO.
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Thiocyano (thiocyanato): -SCN.
Isothiocyano (isothiocyanato): -NCS.
Sulfhydryl (thiol, mercapto): -SH.
Thioether (sulfide): -SR, wherein R is a thioether
substituent, for example, a C1_~ alkyl group (also referred to
as a Cl_~ alkylthio group) , a C3_zo heterocyclyl group, or a CS_zo
aryl group, preferably a Cl_~ alkyl group. Examples of C1_~
alkylthio groups include, but are not limited to, -5CH3 and
-SCHZCH3.
Disulfide: -SS-R, wherein R is a disulfide substituent, for
example, a C1_~ alkyl group, a C3_zo heterocyclyl group, or a CS_zo
aryl group, preferably a C1_~ alkyl group (also referred to
herein as C1_~ alkyl disulfide) . Examples of C1_~ alkyl
disulfide groups include, but are not limited to, -SSCH3 and
-SSCHZCH3.
Sulfone (sulfonyl): -S(=0)zR, wherein R is a sulfone
substituent, for example, a C1_~ alkyl group, a C3_zo
heterocyclyl group, or a CS_zo aryl group, preferably a C1_~
alkyl group. Examples of sulfone groups include, but are not
limited to, -S (=0) zCH3 (methanesulfonyl, mesyl) , -S (=0) zCF3
(triflyl ) , -S (=0) zCH2CH3, -S (=0) zCqF9 (nonaflyl ) , -S (=0) zCH2CF3
(tresyl), -S(=O)zPh (phenylsulfonyl), 4-methylphenylsulfonyl
(tosyl), 4-bromophenylsulfonyl (brosyl), and 4-nitrophenyl
(nosyl).
Sulfine (sulfinyl, sulfoxide): -S(=O)R, wherein R is a sulfine
substituent, for example, a C1_~ alkyl group, a C3-zo
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heterocyclyl group, or a CS_2o aryl group, preferably a C1_~
alkyl group. Examples of sulfine groups include, but are not
limited to, -S (=0) CH3 and -S (=O) CHZCH3.
5 Sulfonyloxy: -OS(=O)zR, wherein R is a sulfonyloxy substituent,
for example, a C1_~ alkyl group, a C3_zo heterocyclyl group, or a
CS_ZO aryl group, preferably a C1_~ alkyl group. Examples of
sulfonyloxy groups include, but are not limited to, -OS(=O)zCH3
and -OS (=O) zCH2CH3.
Sulfinyloxy: -OS(=0)R, wherein R is a sulfinyloxy substituent,
for example, a C1_~ alkyl group, a C3_zo heterocyclyl group, or a
CS_zo aryl group, preferably a C1_~ alkyl group. Examples of
sulfinyloxy groups include, but are not limited to, -OS(=0)CH3
and -OS (=O) CHZCH3.
Sulfamino: -NR1S(=0)zOH, wherein R1 is an amino substituent, as
defined for amino groups. Examples of sulfamino groups
include, but are not limited to, -NHS(=0)zOH and
-N (CH3) S (=0) zOH.
Sulfonamino: -NR1S(=0)zR, wherein R1 is an amino substituent,
as defined for amino groups, and R is a sulfonamino
substituent, for example, a C1_~ alkyl group, a C3_zo
heterocyclyl group, or a CS_zo aryl group, preferably a C1_7
alkyl group. Examples of sulfonamino groups include, but are
not limited to, -NHS (=0) zCH3 and -N (CH3) S (=0) zC6Hs.
Sulfinamino: -NR1S(=0)R, wherein R1 is an amino substituent, as
defined for amino groups, and R is a sulfinamino substituent,
for example, a C1_~ alkyl group, a C3_zo heterocyclyl group, or a
Cs_zo aryl group, preferably a C1_~ alkyl group. Examples of
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sulfinamino groups include, but are not limited to, -NHS(=O)CH3
and -N (CH3) S (=0) C6H5.
Sulfamyl : -S (=0) NRlRz, wherein R1 and Rz are independently amino
substituents, as defined for amino groups. Examples of
sulfamyl groups include, but are not limited to, -S(=O)NHz,
-S (=0) NH (CH3) , -S (=0) N (CH3) z, -S (=0) NH (CHZCH3) , -S (=O) N (CHZCH3)
z.
and -S(=0)NHPh.
Sulfonamino: -NR1S(=0)zR, wherein R1 is an amino substituent,
as defined for amino groups, and R is a sulfonamino
substituent, for example, a C1_~ alkyl group, a C3_zo
heterocyclyl group, or a CS-zo aryl group, preferably a C1_~
alkyl group. Examples of sulfonamino groups include, but are
not limited to, -NHS (=O) zCH3 and -N (CH3) S (=0) zC6H5. A special
class of sulfonamino groups are those derived from sultams -
in these groups one of R1 and R is a CS_2o aryl group,
preferably phenyl, whilst the other of R1 and R is a bidentate
group which links to the CS_zo aryl group, such as a bidentate
group derived from a C1_~ alkyl group. Examples of such groups
include, but are not limited to:
0 0
S
N
2,3-dihydro-tenzo[dJisothiazole-1,1-dioxide-2-yl
-N
I
0 ~S\
0
2 5 1.3-dihydro-benzo(cJisothiazole-2,2-dioxide-1-yl
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O
~~ ~O
,S
N
3,4-dihydro-2H-benzo[a][1,2]thiazine-1,1-dioxide-2-yl
Phosphoramidite : -OP (OR1 ) -NRzz, where R1 and Rz are
phosphoramidite substituents, for example, -H, a (optionally
substituted) C1_~ alkyl group, a C3_zo heterocyclyl group, or a
Cs-zo aryl group, preferably -H, a C1_7 alkyl group, or a CS_zo
aryl group. Examples of phosphoramidite groups include, but
are not limited to, -OP (OCH2CH3) -N (CH3) z, -OP (OCHZCH3) -N (i-Pr) z,
and -OP (OCHZCHzCN ) -N ( i-Pr ) z .
Phosphoramidate: -OP (=0) (OR1) -NRzz, where R1 and Rz are
phosphoramidate substituents, for example, -H, a (optionally
substituted) Cl_~ alkyl group, a C3_zo heterocyclyl group, or a
CS_zo aryl group, preferably -H, a C1_7 alkyl group, or a CS_zo
aryl group. Examples of phosphoramidate groups include, but
are not limited to, -OP (=0) (OCHzCH3) -N (CH3) z, -OP (=O) (OCHZCH3) -
N ( i-Pr ) z, and -OP (=0) ( OCH2CHzCN ) -N ( i-Pr ) z .
In many cases, substituents may themselves be substituted.
For example, a C1_~ alkoxy group may be substituted with, for
example, a C1_~ alkyl (also referred to as a C1_~ alkyl-Cl_~alkoxy
group), for example, cyclohexylmethoxy, a C3_zo heterocyclyl
group (also referred to as a C5_zo aryl-C1_7 alkoxy group) , for
example phthalimidoethoxy, or a C5_zo aryl group (also referred
to as a C5-zoaryl-C1_~alkoxy group) , for example, benzyloxy.
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CS_~ ring
The CS_~ ring in R3 has at least two carbon-carbon double bond,
by virtue of its fusion to a benzene or pyridine ring and a
CS_ZO carboaryl group. If the C5_2o carboaryl group contains a
nitrogen ring atom, this does not form part of the CS_~ ring.
The same applies to the nitrogen ring atom of the pyridyl
group.
Thus, the C5_~ ring may be a CS_~ sulphur containing
heterocycle, a CS_~ oxygen heterocycle, a CS_~ nitrogen
containing heterocycle or a CS_~ cyclic group containing at
least 5 carbon ring atoms.
The second bridge group may typically be a single bond
(resulting in a CS ring), or have 1 or 2 atoms in a chain
(resulting in C6 and C~ rings respectively), which atoms are
usually selected from C, S, 0 and N, with substitution as
appropriate.
CS_~ sulphur containing heterocycle
The C5_7 sulphur containing heterocycle in R3 will have at least
two carbon-carbon double bonds, by virtue of its fusion to a
benzene or pyridine ring and a CS-2o carboaryl group. Examples
of relevant CS_~ sulphur containing heterocycles include, but
are not limited to:
/S
thiophene
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S S S S S
c6: ~ ~ ~ ~
O S N
H
O
thiaine 4-thiaoxaine 1,4-dithiaine 4H-4-azathiaine 4-oxothiaine
S S S S
Co
O N
H
4,5-dihydrothiaepine thiaepine 4-oxa-5-hydrothiaepine 4H-4-aza-5-
hydrothiaepine
S S S
N- I S
O
4-azathiaepine 4-oxo-5-hydrothiaepine 5-hydro-1,4-dithiaepine
The CS_~ sulphur containing heterocycle may be substituted
(when possible) by the substituent group listed above.
The groups shown above may in particular be substituted on the
sulphur atom in the first bridge group by one or two oxo (=0)
groups.
C5_~ oxygen containing heterocycle
The CS_7 oxygen containing heterocycle in R3 will have at least
two carbon-carbon double bonds, by virtue of its fusion to a
benzene or pyridine ring and a CS_2o carboaryl group. Examples
of relevant CS_~ oxygen containing heterocycles include, but
are not limited to:
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/O
furan
O O O O O
C6:
O S
O
4H-pyran 1,4-dioxin 4-thiaoxaine 4H-1,4-Oxazine 4-pyrone
(p-Isoxazine)
5
O O O O
O N
H
4,5-dihydrooxaepine oxaepine 5-hydro-1,4-dioxaepine 4H-4-aza-5-hydro-oxaepine
O O O
N S
O
4-azaoxaepine 4-oxo-4,5-hydrooxaepine 4-thia-5-hydro-oxaepine
The C5-7 oxygen containing heterocycle may be substituted (when
possible) by the substituent groups listed above.
C5-~ nitrogen containing heterocycle
The CS_~ nitrogen containing heterocycle in R3 will have at
least two carbon-carbon double bonds, by virtue of its fusion
to a benzene or pyridine ring and a CS_ZO carboaryl group.
Examples of relevant CS_~ nitrogen containing heterocycles
include, but are not limited to (illustrated with RN - H):
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H
/N
1 H-Pyrrole
N N N N H
N
C6:
O S N S
H I I
1,4-Dihydro-pyridine 4H-[1,4]Oxazine 4H-[1,4]Thiazine 1,4-Dihydro-pyrazine O
4H-[1,4)Thiazine 1-oxide
N
N N N H
c,: I 1 I 1 I 1 I 1
O N
H
4,5-Dihydro-1H-azepine 1H-Azepine 4,7-Dihydro-[1,4]oxazepine 4,5-Dihydro-1H-
[1,4)diazepine
N N N
/ 1 / 1 I 1
N S
O
1H-[1,4]Diazepine 1,5-Dihydro-azepin-4-one 4,7-Dihydro-[1,4]thiazepine
The CS-~ nitrogen containing heterocycle may be substituted
(when possible) by the substituent group listed above. In
particular, the nitrogen atom in the first bridge group may be
substituted by RN.
CS-~ cyclic group containing at least 5 carbon ring atoms
The C5-~ cyclic group containing at least 5 carbon ring atoms
in R3 will have at least two carbon-carbon double bonds, by
virtue of being fused to a benzene or pyridine ring and a CS_Zo
carboaryl group. Examples of relevant CS_7 cyclic group
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containing at least 5 carbon ring atoms include, but are not
limited to:
Cyclopenta-1,3-diene
cs: I I I I I I I I IsJ
Si N~ n
H O
Cyclohexa-1,4-diene 4H-Pyran 4H-Thiopyran 1,4-Dihydro-pyridine 4H-Thiopyran 1-
oxide
C,: / ~ / ~
O H
Cyclohepta-1,4-diene Cyclohepta-1,3,5-triene 2,5-Dihydro-oxepine 2,5-Dihydro-
1H-azepine
N- ~ S
O
4H-Azepine Cyclohepta-2,5-dienone 2,5-Dihydro-thiepine
The CS-7 cyclic group containing at least 5 carbon ring atoms
may be substituted (when possible) by the substituent group
listed above.
Possible R3 Structures
Accordingly, when the phenyl or pyridyl group is linked to a
C5_2o carboaryl group, R3 can be of the following structure,
wherein the phenyl or pyridyl group and the CS-zo carboaryl
group are illustrated as benzene rings, without being limited
thereto, and where X may be 0, S, S(=O), S(=O)2, NRN and
CRclRc2
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/ \
\ /
X
/ /
\ \
X
/ / / S / / O /
\ \ ~ \ \ ~ \
X X X
H O
/ N / / /
\ \ I \
X ~X
O O
/ ~ / ~ /
X ~ ~ X ~ ~ X
S S O p
/ 1 ~ / , ~ / 1 ~ /
X \ ~ X \ ~ X \ ~ ' X \
N- =N N
N
1 ~ / ~ ~ / 1
X \ ~ X \ ~ X ~ ' X
with substitution as appropriate on the above core structures
When the phenyl or pyridyl group is linked to a CS-2o carboaryl
group in which one aromatic carbon ring atom has been replaced
by an aromatic nitrogen ring atom, then R3 can be any of the
structures shown above, where the benzene ring represents a
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CS_ZO carboaryl group containing a nitrogen ring atom, for
example:
/ ~N
\ X /
~/
\ N w X
N~ / N S / / O
\ \ I \ w ~ \
X X N X
H O
/ N / / / /
N~ \ \ ~ ~ \
X ~N X
where X is as defined above.
15
If the first group in R3 is a pyridyl group, rather than the
phenyl group as illustrated above, the nitrogen ring atom may
be at any available position of the ring.
The first bridge may be situated at any possible position of
the phenyl group in R3 and the optional second bridge group may
be situated on either adjacent atom of the phenyl group (if
possible). Therefore, the resulting R3 group (as a whole) may
be a radical at a number of possible positions on the benzene
ring bound to the central moiety, for example the following
possible R3 group (unsubstituted xanthenyl):
4
\ O \3
/ / 2
1
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may be a radical at the 1, 2, 3 or 4 positions.
Includes Other Forms
5 Included in the above are the well known ionic, salt, solvate,
and protected forms of these substituents. For example, a
reference to carboxylic acid (-COOH) also includes the anionic
(carboxylate) form (-COO-), a salt or solvate thereof, as well
as conventional protected forms. Similarly, a reference to an
10 amino group includes the protonated form (-N+HRlRz), a salt or
solvate of the amino group, for example, a hydrochloride salt,
as well as conventional protected forms of an amino group.
Similarly, a reference to a hydroxyl group also includes the
anionic form (-0-), a salt or solvate thereof, as well as
15 conventional protected forms of a hydroxyl group.
Isomers, Salts, Solvates, Protected Forms, and Prodruqs
Certain compounds may exist in one or more particular
geometric, optical, enantiomeric, diasteriomeric, epimeric,
20 stereoisomeric, tautomeric, conformational, or anomeric forms,
including but not limited to, cis- and trans-forms; E- and Z-
forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and
meso-forms; D- and L-forms; d- and 1-forms; (+) and (-) forms;
keto-, enol-, and enolate-forms; syn- and anti-forms;
25 synclinal- and anticlinal-forms; a- and (3-forms; axial and
equatorial forms; boat-, chair-, twist-, envelope-, and
halfchair-forms; and combinations thereof, hereinafter
collectively referred to as "isomers" (or "isomeric forms").
30 Note that, except as discussed below for tautomeric forms,
specifically excluded from the term "isomers", as used herein,
are structural (or constitutional) isomers (i.e. isomers which
differ in the connections between atoms rather than merely by
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the position of atoms in space). For example, a reference to
a methoxy group, -OCH3, is not to be construed as a reference
to its structural isomer, a hydroxymethyl group, -CHZOH.
Similarly, a reference to ortho-chlorophenyl is not to be
construed as a reference to its structural isomer, meta-
chlorophenyl. However, a reference to a class of structures
may well include structurally isomeric forms falling within
that class (e. g., C1_? alkyl includes n-propyl and iso-propyl;
butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl
includes ortho-, meta-, and para-methoxyphenyl).
The above exclusion does not pertain to tautomeric forms, for
example, keto-, enol-, and enolate-forms, as in, for example,
the following tautomeric pairs: keto/enol (illustrated below),
imine/enamine, amide/imino alcohol, amidine/amidine,
nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxyazo, and
nitro/aci-nitro.
O \ OOH H+_ \ /O_
\ /C C\ H+ /C C\
keto enol enolate
Note that specifically included in the term "isomer" are
compounds with one or more isotopic substitutions. For
example, H may be in any isotopic form, including 1H, ZH (D),
and 3H (T) ; C may be in any isotopic form, including 12C, isC,
and 19C; 0 may be in any isotopic form, including 160 and 180;
and the like.
Unless otherwise specified, a reference to a particular
compound includes all such isomeric forms, including (wholly
or partially) racemic and other mixtures thereof. Methods for
the preparation (e. g. asymmetric synthesis) and separation
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(e. g., fractional crystallisation and chromatographic means)
of such isomeric forms are either known in the art or are
readily obtained by adapting the methods taught herein, or
known methods, in a known manner.
Unless otherwise specified, a reference to a particular
compound also includes ionic, salt, solvate, and protected
forms of thereof, for example, as discussed below.
It may be convenient or desirable to prepare, purify, and/or
handle a corresponding salt of the active compound, for
example, a pharmaceutically-acceptable salt. Examples of
pharmaceutically acceptable salts are discussed in Berge et
al., 1977, "Pharmaceutically Acceptable Salts", J. Pharm.
Sci., Vol. 66, pp. 1-19.
For example, if the compound is anionic, or has a functional
group which may be anionic (e.g., -COOH may be -COO-), then a
salt may be formed with a suitable ration. Examples of
suitable inorganic rations include, but are not limited to,
alkali metal ions such as Na+ and K+, alkaline earth rations
such as Ca2+ and Mg2+, and other rations such as A13+. Examples
of suitable organic rations include, but are not limited to,
ammonium ion (i.e., NHq+) and substituted ammonium ions (e.g.,
NH3R+, NHzR2+, NHR3+, NRQ+) . Examples of some suitable
substituted ammonium ions are those derived from: ethylamine,
diethylamine, dicyclohexylamine, triethylamine, butylamine,
ethylenediamine, ethanolamine, diethanolamine, piperazine,
benzylamine, phenylbenzylamine, choline, meglumine, and
tromethamine, as well as amino acids, such as lysine and
arginine. An example of a common quaternary ammonium ion is
N (CH3) 9+.
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If the compound is cationic, or has a functional group which
may be cationic (e.g., -NHz may be -NH3+), then a salt may be
formed with a suitable anion. Examples of suitable inorganic
anions include, but are not limited to, those derived from the
following inorganic acids: hydrochloric, hydrobromic,
hydroiodic, sulphuric, sulphurous, nitric, nitrous,
phosphoric, and phosphorous. Examples of suitable organic
anions include, but are not limited to, those derived from the
following organic acids: acetic, propionic, succinic,
glycolic, stearic, palmitic, lactic, malic, pamoic, tartaric,
citric, gluconic, ascorbic, malefic, hydroxymaleic,
phenylacetic, glutamic, aspartic, benzoic, cinnamic, pyruvic,
salicyclic, sulfanilic, 2-acetyoxybenzoic, fumaric,
phenylsulfonic, toluenesulfonic, methanesulfonic,
ethanesulfonic, ethane disulfonic, oxalic, pantothenic,
isethionic, valeric, lactobionic, and gluconic. Examples of
suitable polymeric anions include, but are not limited to,
those derived from the following polymeric acids: tannic acid,
carboxymethyl cellulose.
It may be convenient or desirable to prepare, purify, and/or
handle a corresponding solvate of the active compound. The
term "solvate" is used herein in the conventional sense to
refer to a complex of solute (e.g. active compound, salt of
active compound) and solvent. If the solvent is water, the
solvate may be conveniently referred to as a hydrate, for
example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.
It may be convenient or desirable to prepare, purify, and/or
handle the active compound in a chemically protected form.
The term "chemically protected form", as used herein, pertains
to a compound in which one or more reactive functional groups
are protected from undesirable chemical reactions, that is,
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are in the form of a protected or protecting group (also known
as a masked or masking group or a blocked or blocking group).
By protecting a reactive functional group, reactions involving
other unprotected reactive functional groups can be performed,
without affecting the protected group; the protecting group
may be removed, usually in a subsequent step, without
substantially affecting the remainder of the molecule. See,
for example, Protective Groups in Organic Synthesis (T. Green
and P. Wuts, Wiley, 1999).
For example, a hydroxy group may be protected as an ether (-
OR) or an ester (-OC(=0)R), for example, as: a t-butyl ether;
a benzyl, benzhydryl (diphenylmethyl), or trityl
(triphenylmethyl) ether; a trimethylsilyl or
t-butyldimethylsilyl ether; or an acetyl ester (-OC(=0)CH3,
-OAc ) .
For example, an aldehyde or ketone group may be protected as
an acetal or ketal, respectively, in which the carbonyl group
(>C=O) is converted to a diether (>C(OR)2), by reaction with,
for example, a primary alcohol. The aldehyde or ketone group
is readily regenerated by hydrolysis using a large excess of
water in the presence of acid.
For example, an amine group may be protected, for example, as
an amide or a urethane, for example, as: a methyl amide
(-NHCO-CH3); a benzyloxy amide (-NHCO-OCHZC6H5, -NH-Cbz); as a
t-butoxy amide (-NHCO-OC(CH3)3, -NH-Boc); a 2-biphenyl-2-
propoxy amide (-NHCO-OC (CH3) ZC6HqC6H5, -NH-Bpoc) , as a 9-
fluorenylmethoxy amide (-NH-Fmoc), as a 6-nitroveratryloxy
amide (-NH-Nvoc), as a 2-trimethylsilylethyloxy amide (-NH-
Teoc), as a 2,2,2-trichloroethyloxy amide (-NH-Troc), as an
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allyloxy amide (-NH-Alloc), as a 2(-phenylsulphonyl)ethyloxy
amide (-NH-Psec); or, in suitable cases, as an N-oxide (>NO~).
For example, a carboxylic acid group may be protected as an
5 ester for example, as: an C1_~ alkyl ester (e. g. a methyl ester;
a t-butyl ester) ; a C1_~ haloalkyl ester (e . g. , a C1_~
trihaloalkyl ester) ; a triCl_~ alkylsilyl-C1_~ alkyl ester; or a
CS-ZO aryl-C1_~ alkyl ester (e.g. a benzyl ester; a nitrobenzyl
ester); or as an amide, for example, as a methyl amide.
For example, a thiol group may be protected as a thioether
(-SR), for example, as: a benzyl thioether; an acetamidomethyl
ether (-S-CHZNHC (=O) CH3) .
It may be convenient or desirable to prepare, purify, and/or
handle the active compound in the form of a prodrug. The term
"prodrug", as used herein, pertains to a compound which, when
metabolised (e. g. in vivo), yields the desired active
compound. Typically, the prodrug is inactive, or less active
than the active compound, but may provide advantageous
handling, administration, or metabolic properties.
For example, some prodrugs are esters of the active compound
(e. g. a physiologically acceptable metabolically labile
ester). During metabolism, the ester group (-C(=O)OR) is
cleaved to yield the active drug. Such esters may be formed
by esterification, for example, of any of the carboxylic acid
groups (-C(=O)OH) in the parent compound, with, where
appropriate, prior protection of any other reactive groups
present in the parent compound, followed by deprotection if
required. Examples of such metabolically labile esters
include those wherein R is C1_~ alkyl (e.g. -Me, -Et) ; C1_~
aminoalkyl (e. g. aminoethyl; 2-(N,N-diethylamino)ethyl;
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2-(4-morpholino)ethyl); and acyloxy-C1_~ alkyl (e. g.
acyloxymethyl; acyloxyethyl; e.g. pivaloyloxymethyl;
acetoxymethyl; 1-acetoxyethyl; 1-(1-methoxy-1-methyl)ethyl-
carbonxyloxyethyl; 1-(benzoyloxy)ethyl; isopropoxy-
carbonyloxymethyl; 1-isopropoxy-carbonyloxyethyl; cyclohexyl-
carbonyloxymethyl; 1-cyclohexyl-carbonyloxyethyl;
cyclohexyloxy-carbonyloxymethyl; 1-cyclohexyloxy-
carbonyloxyethyl; (4-tetrahydropyranyloxy) carbonyloxymethyl;
1-(4-tetrahydropyranyloxy)carbonyloxyethyl;
(4-tetrahydropyranyl)carbonyloxymethyl; and
1-(4-tetrahydropyranyl)carbonyloxyethyl).
Also, some prodrugs are activated enzymatically to yield the
active compound, or a compound which, upon further chemical
reaction, yields the active compound. For example, the
prodrug may be a sugar derivative or other glycoside
conjugate, or may be an amino acid ester derivative.
Further Preferences
The following preferences may be different for different
aspects of the present invention, and may be combined
together.
In compounds of formula I, it is preferred that P is 0 and Q
is CH, i.e. that the compound is of formula Ia.
Y is preferably 0.
In formula h, when R1 and R2 form, along with the nitrogen atom
to which they are attached, a heterocyclic ring having from 4
to 8 atoms, this may form part of a C9_ZO heterocyclyl group
defined above (except with a minimum of 4 ring atoms), which
must contain at least one nitrogen ring atom. It is preferred
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that R1 and RZ form, along with the nitrogen atom to which they
are attached, a heterocyclic ring having 5, 6 or 7 atoms, more
preferably 6 ring atoms.
Single rings having one nitrogen atom include azetidine,
azetidine, pyrrolidine (tetrahydropyrrole), pyrroline (e. g.,
3-pyrroline, 2,5-dihydropyrrole), 2H-pyrrole or 3H-pyrrole
(isopyrrole, isoazole), piperidine, dihydropyridine,
tetrahydropyridine, and azepine; two nitrogen atoms include
imidazolidine, pyrazolidine (diazolidine), imidazoline,
pyrazoline (dihydropyrazole), and piperazine; one nitrogen and
one oxygen include tetrahydrooxazole, dihydrooxazole,
tetrahydroisoxazole, dihydroisoxazole, morpholine,
tetrahydrooxazine, dihydrooxazine, and oxazine; one nitrogen
and one sulphur include thiazoline, thiazolidine, and
thiomorpholine.
Preferred rings are those containing one heteroatom in
addition to the nitrogen, and in particular, the preferred
heteroatoms are oxygen and sulphur. Thus preferred groups
include morpholino, thiomorpholino, thiazolinyl. Preferred
groups without a further heteroatom include pyrrolidino.
The most preferred groups are morpholino and thiomorpholino.
As mentioned above, these heterocyclic groups may themselves
be substituted; a preferred class of substituent is a C1-~
alkyl group. When the heterocyclic group is morpholino, the
substituent group or groups are preferably methyl or ethyl,
and more preferably methyl. A sole methyl substituent is most
preferably in the 2 position.
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As well as the single ring groups listed above, rings with
bridges or cross-links are also envisaged. Examples of these
types of ring where the group contains a nitrogen and an
oxygen atom are:
O O
O
-N~ -N/~O N NJ
These are named 8-oxa-3-aza-bicyclo[3.2.1]oct-3-yl, 6-oxa-3-
aza-bicyclo[3.1.0]hex-3-yl, 2-oxa-5-aza-bicyclo[2.2.1]kept-5-
yl, and 7-oxa-3-aza-bicyclo[4.1.0]kept-3-yl, respectively.
In R3, the phenyl or pyridyl group is preferably a phenyl
group.
R~1 and R~2 are preferably H.
RN is preferably H, or an ester.
Preferred substituents of the phenyl or pyridyl ring in R3
include, but are not limited to, halo, hydroxy, C1-~ alkyl, C1_~
alkoxy, acyl, acyloxy, amino, nitro, cyano, thiol and C1_7
alkylthio, with halo and hydroxy being most preferred.
Preferred substituents of the phenyl or pyridyl ring or the CS_
zo carboaryl group in R3 also include, but are not limited to,
acylamido, sulfonamino, ether, ester, amido, amino and acyl.
In the acylamido group, the amide substituent is preferably
hydrogen, and the acyl substituent is preferably selected from
ester (where the ester substituent is alkyl or aryl), C1_7
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alkyl (optionally substituted by ether, ester, CS-zo aryl,
acyloxy, amino and heterocyclyl), C5-zo aryl (optionally
substituted by alkoxy, alkyl, alkoxy, ester and CS_2o aryl) and
C3-zo heterocylyl (optionally substitued by acyl).
A particularly preferred acyl susbtituent on the acylamido
group is of formula III:
R3
I
*~N~R4 (III)
wherein n is 1 to 4, preferably 1 or 2, and R3 and Rq are
independently hydrogen, an optionally substituted C1_~ alkyl
group, C3-zo heterocyclyl group, or CS-zo aryl group, or may
together form, along with the nitrogen atom to which they are
attached, an optionally substituted heterocyclic ring having
from 4 to 8 ring atoms.
In the sulfonamino group, the amino substituent is preferably
hydrogen and the sulfonamino substituent selected from C1_~
alkyl and CS-zo aryl.
In the ether group, the ether substituent is preferably C1_~
alkyl (optionally substituted by amino, C3-zo heterocyclyl,
thioether and CS-zo aryl). A particularly preferred ether
substituent is of formula III (defined above).
In the amido group, the amido substituents are preferably
independently selected from hydrogen and C1_7 alkyl (optionally
substituted by C3-zo heterocyclyl, CS_ZO aryl and amino) . A
particularly preferred amido substituent is of formula III
(defined above).
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In the acyl group, the acyl substituent is preferably C3_Zo
heterocyclyl.
These substituents are preferably either para to the radical
5 position in the phenyl or pyridyl group, and when the first
bridge group is ortho the radical position in the phenyl or
pyridyl group, para to the first bridge group in the CS_ZO aryl
group (especially when that group is phenyl).
10 Preferred structures for R3 include, but are not limited to the
following 'core' groups, which may bear substitution at
appropriate positions, where * indicates the preferred radical
position (which is typically adjacent the first bridge group
on the phenyl group):
\ \ \ ~ ~ ~ \ ~ ~ \
X ~ X \* X
H
/ S \ / ~ \ / N \
/
X * X * \
X
X
with the most preferred core structures being:
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\ \ \ S \ ~ S \
/ ~ / ~ / / ~ / ~ /
wS S ~ O
H
\ N \ / / \
/ ( / \ \ ~ / ~ I /
S ~ S ~ S
Particularly preferred R3 groups include:
O R ~ ~O
~ S
R- 'NH O~ ~NH OR
O O
R- -N R I \ S
\ ~ \ / ~ /
/ S / S
wherein R stands for the appropriate substituent group, as
defined above.
Acronyms
For convenience, many chemical moieties are represented using
well known abbreviations, including but not limited to, methyl
(Me), ethyl (Et), n-propyl (nPr), iso-propyl (iPr), n-butyl
(nBu), tert-butyl (tBu), n-hexyl (nHex), cyclohexyl (cHex),
phenyl (Ph), biphenyl (biPh), benzyl (Bn), naphthyl (naph),
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methoxy (Me0), ethoxy (Et0), benzoyl (Bz), acetyl (Ac), 1,3-
bis(diphenylphosphino) propane (dppf).
For convenience, many chemical compounds are represented using
well known abbreviations, including but not limited to,
methanol (MeOH), ethanol (EtOH), iso-propanol (i-PrOH), methyl
ethyl ketone (MEK), ether or diethyl ether (Et20), acetic acid
(AcOH), dichloromethane (methylene chloride, DCM),
trifluoroacetic acid (TFA), dimethylformamide (DMF),
tetrahydrofuran (THF), and dimethylsulfoxide (DMSO).
Svnthesis Routes
Compounds according to the first aspect of the invention, of
formula Ia, where Y=0, may be synthesised by the coupling of a
2-chloro-6-amino-pyran-4-one to an appropriate arylboronic
acid or arylboronate ester using a palladium catalysed
coupling reaction, e.g. Suzuki coupling. Compounds where Y=S
can be derived from the corresponding compound where Y=0.
Synthesis of 2-chloro-6-amino-pyran-4-ones
These may be synthesised by the following route:
CI CI O O O
CI
CI ~ CI R'
a
O O ( ) ~ (b) CI N2 (~) ' I ~ ~ R~
O~O R CI O N
Rz
(1)
In step (a) CClq is added across the carbon-carbon double bond
of diketene by free-radical addition to yield 4-chloro-
4(2,2,2,-trichloro-ethyl)-oxetan-2-one (1). Suitable
initiators include peroxide, such as BCHPO ((bis-4-t-
butylcyclohexyl)peroxydicarbonate).
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In step (b), the amine R1RZNH opens the lactone ring by
nucleophilic attack at the carbonyl centre. The oxy anion
generated then displaces the chlorine atom on the a-carbon to
give rise to a (3-keto-amide intermediate. Further elimination
of HC1 finally give the 5,5-dichloro-1-amino-pent-4-ene-1,3-
dione. Suitable conditions for this step include inorganic
base such as sodium hydrogen carbonate and solvent such as dry
dichloromethane.
In step (c), ring closure takes place by displacement of one
of the 5-chloro groups by the oxygen of the amide moiety to
form the pyran-4-one ring, which reaction is catalysed by a
Lewis acid, such as perchloric acid.
Arylboronic Acids and Arylboronate esters
Some appropriate arylboronic acids and arylboronate esters are
commercially available. Other appropriate arylboronic acids
and arylboronate esters may be synthesised by using one of the
following routes, in which the starting materials are
commercially available or readily synthesised. For example, a
synthesis route to thioxanthenone is described in Archer, S.,
et al., J. Med. Chem., 25, 220-227, 1982, and the conversion
of thioxanthenone to thiothanxene is described in Mlotkowska,
B.L., et al., J. Heterocyclic Chem., 28, 731-736, 1991. Other
routes are shown in the examples, and include routes where the
central CS_~ ring is synthesised by ring closure from an
appropriate carboxylic acid, optionally followed by reduction
of the remaining keto group.
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Synthesis of aryl boronate esters
X
a
( ) \ X= TfO, Br, I
R R
(a): PdCl2dppf, dppf, Pinacol diborane, KOAc
where R is the remainder of the R3 group
Aryl boronate esters may be formed by Pd(0)-catalysed cross
coupling reaction of the appropriate aryl triflate or aryl
halide with tetra(alkoxy)diboron, e.g. pinacol diboron.
Suitable conditions include the use of a catalyst, such as
PdClzdppf, extra ligands, such as dppf, potassium acetate as a
base, in a solvent such as dioxane, DMF or DMSO.
Examples of this method are to be found in T Ishiyama, et al.,
Tet. Lett., vol. 38, no. 19, 3447-3450, 1997 and A Giroux, et
al., Tet. Lett., vol. 38, no. 22, 3841-3844, 1997.
Synthesis of aryl boronic acids
HO~B~OH
w (a) I w
/ /
R R
(a)a-BuLi, (Et0)3B
where R is the remainder of the R3 group
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Boronic acids may be generated via lithiation of the aromatic
ring by tert-butyl lithium followed by the reaction of the
anion formed with alkyl borate such as triethyl borate to give
the desired aryl boronic acid.
5
Palladium Catalysed Coupling
The coupling of the arylboronic acid or arylboronate ester to
the 2-chloro-6-amino-pyran-4-one can be carried out using the
normal conditions, e.g. a palladium catalyst (Pd(PPh3)9,
10 Pd (dppf ) C12) and base (Na2C03, NaOCH2CH3, T10H, N (CH2CH3) 3,
K3 PO9 ) .
Compounds according to the first aspect of the invention, of
formula Ib, where Y=0 may be synthesised according to the
15 following method, wherein R represents the rest of R3:
O OH S
R ~ I a R ~ I \ SH
v
OH S OH S
\ SEt ~ , \ N~R1
R \ I ~ R
R
O SEt
1
N~R1 a R
---~ R \ I R2 RZ
O
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In step (a), CSZ is added to the acetophenone derivative, in
the presence of a base, such as potassium tert-butoxide, to
yield a 3-aryl-3-hydroxy-dithioacrylic acid.
In step (b), iodoethane undergoes nucleophliic attack by the
activated thioacid, to yield the ethyl ester. Activation of
the thioacid can be achieved by the use of base, for example,
a mixture of tetrabutylammonium hydrogen sulphate and sodium
hydroxide.
In step (c), an amine displaces the ethyl group, which is
followed in step (d) by reaction of the remaining thio group
with iodoethane (via the tautomeric compound).
The final step (e) is a condensation with ethyl bromoacetate
to yield the ring-closed 4-amino-6-aryl-pyran-2-one.
Conversion of Y from 0 to S
This conversion may be achieved using Lawesson's reagent in an
organic solvent, such as toluene, followed by the appropriate
purification steps. Protection of groups sensitive to
Lawesson's reagent can be carried out before it is used,
followed by deprotection once the pyranthione has been
synthesised.
Use of Compounds of the Invention
The present invention provides active compounds, specifically,
active 2-aryl-6-amino-pyran-4-ones, 2-aryl-6-amino-pyran-4-
thiones, 4-amino-6-aryl-pyran-2-ones and 4-amino-6-aryl-pyran-
2-thiones.
The term ~~active", as used herein, pertains to compounds which
are capable of inhibiting ATM activity, and specifically
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includes both compounds with intrinsic activity (drugs) as
well as prodrugs of such compounds, which prodrugs may
themselves exhibit little or no intrinsic activity.
One assay which may be used in order to assess the ATM
inhibition offered by a particular compound is described in
the examples below.
The present invention further provides a method of inhibiting
ATM in a cell, comprising contacting said cell with an
effective amount of an active compound, preferably in the form
of a pharmaceutically acceptable composition. Such a method
may be practised in vitro or in vivo.
For example, a sample of cells (e.g. from a tumour) may be
grown in vitro and an active compound brought into contact
with said cells in conjunction with agents that have a known
curative effect, and the enhancement of the curative effect of
the compound on those cells observed.
The present invention further provides active compounds which
inhibit ATM activity as well as methods of inhibiting ATM
activity comprising contacting a cell with an effective amount
of an active compound, whether in vitro or in vivo.
The invention further provides active compounds for use in a
method of treatment of the human or animal body. Such a
method may comprise administering to such a subject a
therapeutically-effective amount of an active compound,
preferably in the form of a pharmaceutical composition.
The term "treatment" as used herein in the context of treating
a condition, pertains generally to treatment and therapy,
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whether of a human or an animal (e. g. in veterinary
applications), in which some desired therapeutic effect is
achieved, for example, the inhibition of the progress of the
condition, and includes a reduction in the rate of progress, a
halt in the rate of progress, amelioration of the condition,
and cure of the condition. Treatment as a prophylactic
measure (i.e. prophylaxis) is also included.
The term "therapeutically-effective amount" as used herein,
pertains to that amount of an active compound, or a material,
composition or dosage form comprising an active compound,
which is effective for producing some desired therapeutic
effect, commensurate with a reasonable benefit/risk ratio.
The present inventors have. found that compounds of the present
invention can efficiently repress retroviral vector
transduction in one-step, cell based integration assays
(termed LUCIA) and inhibit HIV-1 infection in 4-day
replication assays at sub-micromolar concentrations. Further,
in contrast to the observations of Daniel et al., where it was
concluded that the effect of ATM on retroviral integration
would only be seen in a DNA-PK-deficient background, this
effect works in the presence of functional DNA-PK activity.
Initial linkage of linear retroviral DNA with host cell
chromosomal DNA is catalysed by viral integrase (IN) and
results in short staggered DNA strand breaks in the host cell
DNA at the site of attachment (Brown, P.O. (1990) Integration
of retroviral DNA. Curr Top Microbiol Immunol, 157, 19-48).
These gapped DNA intermediates are shown to be sensed as sites
of DNA damage by the host cell and repaired by the ATM pathway
to complete the process of integration and allow productive
infection to occur. Compounds of the invention prevent the
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repair of gapped DNA intermediates by the ATM pathway and thus
prevent complete integration of retroviral DNA into the host
genome.
As described above, the invention provides a compound as
defined in the first aspect of the invention for use in the
treatment of retroviral infection and the use of such a
compound in the manufacture of a medicament for use in the
treatment of retroviral infection.
Also provided by the invention is a method of treatment of a
retroviral infection comprising administering a compound as
defined in the first aspect of the invention to an individual
in need thereof.
An exemplary compound of the invention which is shown to be
useful in the treatment of retroviral infection is 2-
Thianthren-1-yl-6-morpholin-4-yl-pyran-4-one (4).
Retroviral mediated diseases which may be treated as described
above include HIV infection and acquired immunodeficiency
syndrome (AIDS) and Human T-cell Leukaemia virus (HTLV)
infection and its associated diseases adult T-cell
leukaemia/lymphoma (ATLL) and tropical spastic
paraparesis/HTLV-1 associated myelopathy (TSP/HAM).
Compounds of the invention may be used in combination with
other retroviral therapies to suppress virus replication, for
example in a 'highly active anti-retroviral therapy' or HAART
treatment.
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The invention provides a pharmaceutical composition comprising
a compound as described herein and one or more other anti-
retroviral agents.
5 The invention also provides a composition comprising a
compound as defined in the first aspect of the invention and
one or more other anti-retroviral agents for treatment of a
retroviral infection and the use of such a composition in the
manufacture of a medicament for use in the treatment of a
10 retroviral infection.
Suitable anti-retroviral agents which inhibit retroviral
replication, for example retroviral protease inhibitors (PI)
such as Sequinavir, Indinavir, Ritonavir and Nelfinavir,
15 nucleoside retroviral reverse transcriptase inhibitors such as
3'-azido-3'deoxythymidine (AZT; Zidovudine), 2', 3'-
Dideoxycytosine (ddC; Zalcitabine), 2', 3'-Dideoxyinosine
(ddI; Didanosine)and 3TC; (Lamivudine), and non-nucleoside
retroviral reverse transcriptase inhibitors such as
20 Nevirapine, Delavirdine and Efavirenz.
Administration
The active compound or pharmaceutical composition comprising
the active compound may be administered to a subject by any
25 convenient route of administration, whether systemically/
peripherally or at the site of desired action, including but
not limited to, oral (e. g. by ingestion); topical (including
e.g. transdermal, intranasal, ocular, buccal, and sublingual);
pulmonary (e. g. by inhalation or insufflation therapy using,
30 e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal;
parenteral, for example, by injection, including subcutaneous,
intradermal, intramuscular, intravenous, intraarterial,
intracardiac, intrathecal, intraspinal,'intracapsular,
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subcapsular, intraorbital, intraperitoneal, intratracheal,
subcuticular, intraarticular, subarachnoid, and intrasternal;
by implant of a depot, for example, subcutaneously or
intramuscularly.
The subject may be a eukaryote, an animal, a vertebrate
animal, a mammal, a rodent (e.g. a guinea pig, a hamster, a
rat, a mouse), murine (e. g. a mouse), canine (e. g. a dog),
feline (e. g. a cat), equine (e. g. a horse), a primate, simian
(e.g. a monkey or ape), a monkey (e.g. marmoset, baboon), an
ape (e.g. gorilla, chimpanzee, orang-utan, gibbon), or a
human.
Formulations
While it is possible for the active compound to be
administered alone, it is preferable to present it as a
pharmaceutical composition (e.g. formulation) comprising at
least one active compound, as defined above, together with one
or more pharmaceutically acceptable carriers, adjuvants,
excipients, diluents, fillers, buffers, stabilisers,
preservatives, lubricants, or other materials well known to
those skilled in the art and optionally other therapeutic or
prophylactic agents.
Thus, the present invention further provides pharmaceutical
compositions, as defined above, and methods of making a
pharmaceutical composition comprising admixing at least one
active compound, as defined above, together with one or more
pharmaceutically acceptable carriers, excipients, buffers,
adjuvants, stabilisers, or other materials, as described
herein.
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The term "pharmaceutically acceptable" as used herein pertains
to compounds, materials, compositions, and/or dosage forms
which are, within the scope of sound medical judgement,
suitable for use in contact with the tissues of a subject
(e. g. human) without excessive toxicity, irritation, allergic
response, or other problem or complication, commensurate with
a reasonable benefit/risk ratio. Each carrier, excipient,
etc. must also be "acceptable" in the sense of being
compatible with the other ingredients of the formulation.
Suitable carriers, excipients, etc. can be found in standard
pharmaceutical texts, for example, Remington's Pharmaceutical
Sciences, 18th edition, Mack Publishing Company, Easton, Pa.,
1990.
The formulations may conveniently be presented in unit dosage
form and may be prepared by any methods well known in the art
of pharmacy. Such methods include the step of bringing into
association the active compound with the carrier which
constitutes one or more accessory ingredients. In general,
the formulations are prepared by uniformly and intimately
bringing into association the active compound with liquid
carriers or finely divided solid carriers or both, and then if
necessary shaping the product.
Formulations may be in the form of liquids, solutions,
suspensions, emulsions, elixirs, syrups, tablets, losenges,
granules, powders, capsules, cachets, pills, ampoules,
suppositories, pessaries, ointments, gels, pastes, creams,
sprays, mists, foams, lotions, oils, boluses, electuaries, or
aerosols.
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Formulations suitable for oral administration (e.g. by
ingestion) may be presented as discrete units such as
capsules, cachets or tablets, each containing a predetermined
amount of the active compound; as a powder or granules; as a
solution or suspension in an aqueous or non-aqueous liquid; or
as an oil-in-water liquid emulsion or a water-in-oil liquid
emulsion; as a bolus; as an electuary; or as a paste.
A tablet may be made by conventional means, e.g., compression
or moulding, optionally with one or more accessory
ingredients. Compressed tablets may be prepared by
compressing in a suitable machine the active compound in a
free-flowing form such as a powder or granules, optionally
mixed with one or more binders (e. g. povidone, gelatin,
acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose);
fillers or diluents (e. g. lactose, microcrystalline cellulose,
calcium hydrogen phosphate); lubricants (e. g. magnesium
stearate, talc, silica); disintegrants (e. g. sodium starch
glycolate, cross-linked povidone, cross-linked sodium
carboxymethyl cellulose); surface-active or dispersing or
wetting agents (e. g. sodium lauryl sulfate); and preservatives
(e. g. methyl p-hydroxybenzoate, propyl p-hydroxybenzoate,
sorbic acid). Moulded tablets may be made by moulding in a
suitable machine a mixture of the powdered compound moistened
with an inert liquid diluent. The tablets may optionally be
coated or scored and may be formulated so as to provide slow
or controlled release of the active compound therein using,
for example, hydroxypropylmethyl cellulose in varying
proportions to provide the desired release profile. Tablets
may optionally be provided with an enteric coating, to provide
release in parts of the gut other than the stomach.
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Formulations suitable for topical administration (e. g.
transdermal, intranasal, ocular, buccal, and sublingual) may
be formulated as an ointment, cream, suspension, lotion,
powder, solution, past, gel, spray, aerosol, or oil.
Alternatively, a formulation may comprise a patch or a
dressing such as a bandage or adhesive plaster impregnated
with active compounds and optionally one or more excipients or
diluents.
Formulations suitable for topical administration in the mouth
include losenges comprising the active compound in a flavoured
basis, usually sucrose and acacia or tragacanth; pastilles
comprising the active compound in an inert basis such as
gelatin and glycerin, or sucrose and acacia; and mouthwashes
comprising the active compound in a suitable liquid carrier.
Formulations suitable for topical administration to the eye
also include eye drops wherein the active compound is
dissolved or suspended in a suitable carrier, especially an
aqueous solvent for the active compound.
Formulations suitable for nasal administration, wherein the
carrier is a solid, include a coarse powder having a particle
size, for example, in the range of about 20 to about 500
microns which is administered in the manner in which snuff is
taken, i.e. by rapid inhalation through the nasal passage from
a container of the powder held close up to the nose. Suitable
formulations wherein the carrier is a liquid for
administration as, for example, nasal spray, nasal drops, or
by aerosol administration by nebuliser, include aqueous or
oily solutions of the active compound.
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Formulations suitable for administration by inhalation include
those presented as an aerosol spray from a pressurised pack,
with the use of a suitable propellant, such as
dichlorodifluoromethane, trichlorofluoromethane, dichoro-
5 tetrafluoroethane, carbon dioxide, or other suitable gases.
Formulations suitable for topical administration via the skin
include ointments, creams, and emulsions. When formulated in
an ointment, the active compound may optionally be employed
10 with either a paraffinic or a water-miscible ointment base.
Alternatively, the active compounds may be formulated in a
cream with an oil-in-water cream base. If desired, the
aqueous phase of the cream base may include, for example, at
least about 30o w/w of a polyhydric alcohol, i.e., an alcohol
15 having two or more hydroxyl groups such as propylene glycol,
butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene
glycol and mixtures thereof. The topical formulations may
desirably include a compound which enhances absorption or
penetration of the active compound through the skin or other
20 affected areas. Examples of such dermal penetration enhancers
include dimethylsulfoxide and related analogues.
When formulated as a topical emulsion, the oily phase may
optionally comprise merely an emulsifier (otherwise known as
25 an emulgent), or it may comprises a mixture of at least one
emulsifier with a fat or an oil or with both a fat and an oil.
Preferably, a hydrophilic emulsifier is included together with
a lipophilic emulsifier which acts as a stabiliser. It is
also preferred to include both an oil and a fat. Together,
30 the emulsifiers) with or without stabilisers) make up the
so-called emulsifying wax, and the wax together with the oil
and/or fat make up the so-called emulsifying ointment base
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which forms the oily dispersed phase of the cream
formulations.
Suitable emulgents and emulsion stabilisers include Tween 60,
Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl
monostearate and sodium lauryl sulphate. The choice of
suitable oils or fats for the formulation is based on
achieving the desired cosmetic properties, since the
solubility of the active compound in most oils likely to be
used in pharmaceutical emulsion formulations may be very low.
Thus the cream should preferably be a non-greasy, non-staining
and washable product with suitable consistency to avoid
leakage from tubes or other containers. Straight or branched
chain, mono- or dibasic alkyl esters such as di-isoadipate,
isocetyl stearate, propylene glycol diester of coconut fatty
acids, isopropyl myristate, decyl oleate, isopropyl palmitate,
butyl stearate, 2-ethylhexyl palmitate or a blend of branched
chain esters known as Crodamol CAP may be used, the last three
being preferred esters. These may be used alone or in
combination depending on the properties required.
Alternatively, high melting point lipids such as white soft
paraffin and/or liquid paraffin or other mineral oils can be
used.
Formulations suitable for rectal administration may be
presented as a suppository with a suitable base comprising,
for example, cocoa butter or a salicylate.
Formulations suitable for vaginal administration may be
presented as pessaries, tampons, creams, gels, pastes, foams
or spray formulations containing in addition to the active
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compound, such carriers as are known in the art to be
appropriate.
Formulations suitable for parenteral administration (e.g. by
injection, including cutaneous, subcutaneous, intramuscular,
intravenous and intradermal), include aqueous and non-aqueous
isotonic, pyrogen-free, sterile injection solutions which may
contain anti-oxidants, buffers, preservatives, stabilisers,
bacteriostats, and solutes which render the formulation
isotonic with the blood of the intended recipient; and aqueous
and non-aqueous sterile suspensions which may include
suspending agents and thickening agents, and liposomes or
other microparticulate systems which are designed to target
the compound to blood components or one or more organs.
Examples of suitable isotonic vehicles for use in such
formulations include Sodium Chloride Injection, Ringer's
Solution, or Lactated Ringer's Injection. Typically, the
concentration of the active compound in the solution is from
about 1 ng/ml to about 10 ug/ml, for example from about 10
ng/ml to about 1 pg/ml. The formulations may be presented in
unit-dose or multi-dose sealed containers, for example,
ampoules and vials, and may be stored in a freeze-dried
(lyophilised) condition requiring only the addition of the
sterile liquid carrier, for example water for injections,
immediately prior to use. Extemporaneous injection solutions
and suspensions may be prepared from sterile powders,
granules, and tablets. Formulations may be in the form of
liposomes or other microparticulate systems which are designed
to target the active compound to blood components or one or
more organs.
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Dosage
It will be appreciated that appropriate dosages of the active
compounds, and compositions comprising the active compounds,
can vary from patient to patient. Determining the optimal
dosage will generally involve the balancing of the level of
therapeutic benefit against any risk or deleterious side
effects of the treatments of the present invention. The
selected dosage level will depend on a variety of factors
including, but not limited to, the activity of the particular
compound, the route of administration, the time of
administration, the rate of excretion of the compound, the
duration of the treatment, other drugs, compounds, and/or
materials used in combination, and the age, sex, weight,
condition, general health, and prior medical history of the
patient. The amount of compound and route of administration
will ultimately be at the discretion of the physician,
although generally the dosage will be to achieve local
concentrations at the site of action which achieve the desired
effect without causing substantial harmful or deleterious
side-effects.
Administration in vivo can be effected in one dose,
continuously or intermittently (e.g. in divided doses at
appropriate intervals) throughout the course of treatment.
Methods of determining the most effective means and dosage of
administration are well known to those of skill in the art and
will vary with the formulation used for therapy, the purpose
of the therapy, the target cell being treated, and the subject
being treated. Single or multiple administrations can be
carried out with the dose level and pattern being selected by
the treating physician.
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In general, a suitable dose of the active compound is in the
range of about 100 ug to about 250 mg per kilogram body weight
of the subject per day. Where the active compound is a salt,
an ester, prodrug, or the like, the amount administered is
calculated on the basis of the parent compound and so the
actual weight to be used is increased proportionately.
wwwrnT tea
The following examples are provided solely to illustrate the
present invention and are not intended to limit the scope of
the invention, as described herein.
In these examples, reference is made to the following figures.
Figure 1 shows that compound 4 can sensitise cells to ionising
radiation. The o survival of HeLa cells was measured with
increasing ionising radiation, in the absence of compound 4
(~), and at two different concentrations of compound 4, 0.5 ~M
and 2 uM (~).
Figure 2 shows that compound 4 can sensitise cells to
etopside. The o survival of LoVo cells was measured with
increasing concentrations of etopside, in the absence of
compound 4 (~), and in the presence of lOUM of compound 4
Figure 3 shows that compound 4 can sensitise cells to
camptothecin. The °s survival of LoVo cells was measured with
increasing concentrations of camptothecin, in the absence of
compound 4 (~), and in the presence of lOUM of compound 9
Figure 4 shows that compound 4 can sensitise cells to
doxorubicin. The o survival of LoVo cells was measured with
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increasing concentrations of doxorubicin, in the absence of
compound 4 (~), and in the presence of lOUM of compound 4
Figure 5 shows that compound 4 can inhibit recombinant
5 retroviral vector infections. The inhibition of retroviral
transduction by the ATM inhibitor Compound 9 was assessed by
performing HIV-1 based LUCIA on Jurkat T-cells in the presence
of increasing concentrations of compound 4 (~). Data are
presented as transduction efficiency (luciferase signal)
10 relative to untreated control cells. The ICSO concentration
for HIV-1 infections by compound 4 is around 1 uM. Drug
cytotoxicity (O) was determined by MTS formazan dye reduction
assays and data are presented as the percentage of viable
cells remaining after drug treatment. No significant
15 cytotoxicity was observed over the concentration range of
Compound 4 tested.
Figure 6 shows that Compound 4 does not inhibit HIV-1 RT. The
inhibition of HIV-1 RT was assessed by performing
20 chemilluminescent HIV-1 reverse transcriptase assays in the
presence of increasing amounts of compound 4 (~). No
significant anti-RT activity for compound 4 is observed over
the concentration range used. Control RT inhibition using
nevirapine (~) is also shown.
Figure 7 shows that Compound 4 acts synergistically with AZT
to inhibit HIV-1 infections. HIV-1 based LUCIA was performed
on HeLa cells with increasing concentrations of Compound 4 in
the absence (~) or presence of 0.1 uM (0), 0.4 pM (*) or 1.2
~M (0) AZT. Data are presented as transduction efficiency (as
determined by luciferase activity) relative to untreated
control cells. The combined presence of both Compound 4 and
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AZT shows enhanced anti-HIV activity when compared to each
drug alone.
Figure 8 shows that Compound 4 inhibits HIV-1 replication. 9-
day HIV-1 replication assays were performed on C1866 cells in
the presence of increasing concentrations of Compound 4 (~) or
AZT (O). HIV-1 titres were quantified by p24 antigen ELISA
and data are shown as the percentage of HIV-1 p24 in cell-free
supernatants relative to untreated control cells. (A)
Replication assays performed using wild type HIV-1 strain
(HIV-lHxs2 wt). (B) Replication assays performed using an AZT
resistant HIV-1 strain (HIV-lHxs2 AZTres). Compound 4 inhibits
HIV-1 replication equally well in both wild-type and AZT
resistant HIV-1 strains. (C) Control drug cytotoxicity (0)
was determined by XTT dye reduction assays. Data are
presented as the percentage of viable cells remaining after
drug treatment. No significant cytotoxicity was observed over
the effective Compound 4 concentration range shown to inhibit
HIV-1 replication.
A) Chemical Examples
General Experimental Methods
Thin layer chromatography was carried out using Merck
Kieselgel 60 FZS9 glass backed plates. The plates were
visualized by the use of a UV lamp (254 nm). Silica gel 60
(particle sizes 40-63 ~) supplied by E.M.Merck was employed
for flash chromatography. 1H NMR spectra were recorded at 300
MHz on a Bruker DPX-300 instrument. Chemical shifts were
referenced to tetramethylsilane.
Purification and identification of libraries samples
The samples were purified on Gilson LC units.
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Mobile phase A - 0.1% aqueous TFA, Mobile phase B -
Acetonitrile, Flow rate 6 ml/min., Gradient - typically
starting at 90o A/l0o B for one minute, rising to 97o B after
15 minutes, holding there for 2 minutes, then back to the
starting conditions. Column: Jones Chromatography Genesis 4u
C18 column, 10 mm x 250 mm. Peak acquisition based on UV
detection at 259 nm.
Mass Specs were recorded on a Finnegan LCQ instrument in
positive ion mode.
Mobile phase A - 0.1°s aqueous formic acid, Mobile phase B -
Acetonitrile, Flow rate 2 ml/min., Gradient - starting at 95%
A/5o B for one minute, rising to 98o B after 5 minutes,
holding there for 3 minutes, then back to the starting
conditions. Column - Phenomenex 5~ Luna C18 column, 4.6 mm x 50 mm
UV detection at 254 nm, PDA detection scanning from 210 to 600 nm.
Mass spectra of Other Compounds
Mass spectra of non-library compounds and building blocks were
recorded on a Micromass ZQ instrument (single quadrupole,
operating in electrospray ionisation mode), using a Waters 600
HPLC Pump and 2700 Autosampler.
Mobile Phase A: 0.1% Formic acid in water, Mobile phase B:
0.1o Formic acid in acetonitrile, Flow rate: 2.0 ml/min.,
Gradient: 5oB to 95oB over 3mins, hold 3mins. Column: Varies,
but always C18 50 mm x 4.6 mm (Currently Genesis C18 4 ~.
Jones Chromatography). PDA detection: Waters 996, scan range
210-400 nm.
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Synthesis of 2-Chloro-6-morpholin-4-yl-pyran-4-one (3)
CI CI O
CI O O
CI CI
O ~CI ~ ' CI 0 N
O O--~ O
O
1 0 2 3
4-Chloro-4-(2,2,2-trichloro-ethyl)-oxetan-2-one (1)
A solution of BCHPO (bis-4-t-butylcyclohexyl)peroxydicarbonate
(11.8 g) and diketene (83.5 ml) in CC19 (300 ml) was added
dropwise over 120 minutes to a refluxing solution of CC19, and
was stirred for a further 1 hour. The resulting pale yellow
solution was cooled and azeotroped with DCM. The resulting
residue was stirred with hexane (3 x 150 ml) for 10 minutes
and the liquor was decanted off through a celite pad. The
filtered liquors were combined and concentrated in vacuo to
give 1 as a pale yellow oil (125.0 g, 52.90).
5,5-Dichloro-1-morpholin-4-yl-pent-4-ene-1,3-dione (2)
Two separate solutions of 1 (62.5 g, 0.26 mmol) and morpholine
(24.0 g, 0.28 mol) in DCM (120 ml) were added simultaneously
to a mixture of NaHC03 (44.0 g, 0.52 mol) in dry DCM (300 ml).
The reaction was maintained at 15°C over 140 minutes with
stirring. The reaction was filtered, washed with DCM (3 x
100 ml) and the combined organic layers were concentrated in
vacuo to a slurry which was then passed through a short silica
pad, and further washed with DCM (4 x 100 ml). The combined
organic layers were concentrated in vacuo, suspended in hexane
(400 ml) and stirred for 1 hour, filtered and dried to give a
cream solid. The solid was suspended in TBME (100 ml), stirred
for 15 minutes, filtered, washed with TBME and dried to give 2
as a white powder (47.8 g, 720). m/z (LC-MS, ESP): 252 (M+
+1 ) .
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2-Chloro-6-morpholin-4-yl-pyran-4-one (3)
To a suspension of 2 (11.3 g, 44.9 mmol) in dioxane was added
perchloric acid (11.4 ml, 0.14 mol) and the reaction was
heated at 90°C under NZ for 1 hour. The reaction was cooled,
neutralised with 2M NaOH (75 ml) and filtered. The aqueous
layer was extracted with DCM (4 x 30 ml) and the organic
layers were combined and dried over MgS09. The organic layer
was further treated with charcoal and filtered through celite.
The dark yellow filtrate was evaporated in vacuo, and the
resulting solid was triturated with hexane (50 ml) and dried
to give 3 (7.3 g, 750) as a light yellow powder. m/z (LC-MS,
ESP) : 216 (M+ +1) . 1H-NMR (300MHz, DMSO-d6) : 3. 3 (t, 4H) , 3.65
(t, 4H) , 5. 4 (d, 1H) , 6.25 (d, 1H) .
Example 1: Synthesis of 2-Thianthren-1-yl-6-morpholin-4-yl-
pyran-4-one (4)
O
o
I I
I~ o
4
2-Chloro-6-morpholin-4-yl-pyran-4-one (3) (863 mg, 4 mmol),
thianthrene-1-boronic acid (1.145 g, 4.4 mmol), and ground
potassium carbonate (1.105 g, 8 mmol) were suspended in
dioxane (10 ml) and degassed (sonication for 5 minutes then
saturated with N2). Pd(PPh3)q (231 mg, 0.2 mmol) was then added
and the reaction mixture was then heated at 90°C for 24 hours
under a vigorous stirring and a NZ atmosphere. The solvent was
removed in vacuo and the residue was then suspended in water
50 ml) and extracted with ethyl acetate (3 x 100 ml). The
organics were combined, washed with saturated brine and dried
over sodium sulphate. The solvent was removed in vacuo and the
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residue was purified by column chromatography (silica; ethyl
acetate: ethanol; 9:1) to give the title compound as a white
solid (70 mg, 4 0) . 1H-NMR (300MHz, DMSO-d6) . 8H = 3.44 (4H, t,
J 5Hz) ; 3.76 (4H, t, J 5Hz) ; 5.57 (1H, d, J 2Hz) ; 6.30 (1H, d,
5 J 2Hz); 7.43 (2H, m); 7.53 (1H, t, 8Hz); 7.66 (3H, m); 8.49
(1H, dd, J land 8 Hz). m/z (LC-MS, ESP) . 396 (M+ +1).
Example 2: Synthesis of 2-Phenoxathiin-4-yl-6-morpholin-4-yl-
pyran-4-one (5)
~O
O NJ
O
5
2-Chloro-6-morpholin-4-yl-pyran-4-one (3)(863 mg, 4 mmol),
phenoxathiin-4-boronic acid (1.07 g, 4.9 mmol), and ground
potassium carbonate (1.1 g, 8 mmol) were suspended in dioxane
(10 ml) and degassed (sonication for 5 minutes then saturated
with NZ). Pd(PPh3)9 (231 mg, 0.2 mmol) was then added and the
reaction mixture was then heated at 90°C for 24 hours under a
vigorous stirring and a Nz atmosphere. The solvent was removed
in vacuo and the residue was then suspended in water (50 ml)
and extracted with ethyl acetate (3 x 50 ml). The organics
were combined, washed with saturated brine and dried over
sodium sulphate. The solvent was removed in vacuo and the
residue was purified by column chromatography (silica; ethyl
acetate: ethanol; 9:1) to give the title compound as a white
solid (620 mg, 41%). 1H-NMR (300MHz, DMSO-d6) . 8 = 3.38 (4H,
t, J 5Hz) ; 3.71 (4H, t, J 5Hz) ; 5.49 (1H, d, J 2Hz) ; 6.49 (1H,
d, J 2Hz); 7.06 (1H, dd, J 1 and 8Hz); 7.26 (9H, m); 7.46 (1H,
dd, J 1.5 and 8Hz); 7.55 (1H, dd, J 1.5 and 8 Hz). m/z (LC-
MS, ESP) . 380 (M+ +1) .
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Example 3: Synthesis of 2-Dibenzofuran-1-yl-6-morpholin-4-yl-
pyran-4-one (6)
~o
D
r
0
6
2-Chloro-6-morpholin-4-yl-pyran-4-one (3)(22 mg, 0.1 mmol), 4-
dibenzofuran-1-boronic acid (28 mg, 0.13 mmol), and caesium
carbonate (65 mg, 0.2 mmol) were suspended in dioxane (0.5 ml)
and degassed (sonication for 5 minutes then saturated with NZ).
Pd(PPh3)9 (5 mg, 0.005 mmol) was then added and the reaction
mixture was then heated at 90°C for 24 hours under a vigorous
stirring and a N2 atmosphere. The reaction mixture was purified
by preparative HPLC to give the title compound (2.1 mg; 6%).
m/z (LC-MS, ESP) : 348 (M+ +1) .
Example 4: Synthesis of 2-Dibenzothiophen-1-yl-6-morpholin-4-
yl-pyran-4-one (7)
~O
0
r
0
2-Chloro-6-morpholin-4-yl-pyran-4-one (3)(740 mg, 3.43 mmol),
dibenzothiophene-1-boronic acid (860 mg, 3.77 mmol), and
ground potassium carbonate (964 mg, 6.86 mmol) were suspended
in dioxane (10 ml) and degassed (sonication for 5 minutes then
saturated with Nz). Pd(PPh3)9 (200 mg, 0.17 mmol) was then
added and the reaction mixture was then heated at 90°C for 24
hours under a vigorous stirring and a NZ atmosphere. The
solvent were removed in vacuo and the residue was then
suspended in water (50 ml) and extracted with ethyl acetate (3
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x 50 ml). The organics were combined, washed with saturated
brine and dried over sodium sulphate. The solvent was removed
in vacuo and the residue was purified by column chromatography
(silica; ethyl acetate: ethanol; 9:1) to give the title
compound as a white solid (80 mg, 60). 1H-NMR (300MHz, DMSO-
d6) : 8H = 3.49 (4H, t, J 5Hz) ; 3.76 (9H, t, J 5Hz) ; 5.53 (1H,
d, J 2Hz) ; 6.63 (1H, d, J 2Hz) ; 7.59 (2H, m) ; 7. 69 (1H, t, J
8Hz); 7.96 (1H, dd, J 1 and 7.5Hz); 8.11 (1H, m); 8.47 (1H,
m); 8.57 (1H, dd, J 1 and 8 Hz). m/z (LC-MS, ESP): 364 (M+ +1).
Example 5: Synthesis of 2-(2-Phenylsulfanyl-phenyl)-6-
morpholin-4-yl-pyran-4-one (9)
(a) 2-phenylsulfido-benzene boronic acid (8)
HO~ ,OH
B
/ S
8
To a cooled (-78°C), stirred solution of diphenyl sulphide
(1.66 ml, 10 mmol) in 30m1 anhydrous THF, was added dropwise
under a nitrogen atmosphere 7 ml t-BuLi. Upon addition of t-
BuLi the solution turned orange then brown. The mixture was
allowed to warm to room temperature and then left stirring for
3 hours. The mixture was cooled (-78°C). Triethyl borate
(2.03 ml, 12 mmol) was added dropwise to the cooled yellow
solution turning the solution lime coloured. During this
addition, the temperature was monitored and not allowed to
rise above -75°C. The solution was then left to warm to room
temperature and left stirring for 2 hours. Water was added to
the reaction mixture and the aqueous were extracted with
diethyl ether. The aqueous layer (pH 14) was acidified to pH
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1 with (1 M HC1) and the product was extracted into diethyl
ether. The organics were dried over magnesium sulphate and the
organics were evaporated off in vacuo, yielding an oily
residue (690 mg, 300), which was used without further
purification.
(b) 2-(2'-Phenylsulfido-phenyl)-6-morpholin-4-yl-pyran-4-one
(9)
~o
0
0
9
2-Chloro-6-morpholin-4-yl-pyran-4-one (3)(582 mg, 2.7 mmol),
2-phenylsulphido-benzene boronic acid (8) (690 g, 3 mmol), and
ground potassium carbonate (819 mg, 5.94 mmol) were suspended
in dioxane (lOml) and degassed (sonication for 5 minutes then
saturated with NZ). Pd(PPh3)4 (156 mg, 0.13 mmol) was then added
and the reaction mixture was then heated at 90°C for 24 hours
under a vigorous stirring and a NZ atmosphere. The solvent was
removed in vacuo and the residue was purified by preparative
HPLC to give the title compound (27mg, 3%). 1H-NMR (300MHz,
DMSO-d6) : 8H = 3.37 (4H, t) ; 3.76 (4H, t) 5.45 (1H, d) ; 6.31
(1H, d); 7.32-7.55 (9H, m). m/z (LC-MS, ESP): 366 (M+ +1).
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Example 6: Synthesis of 2-(1-Fluoro-9-oxo-9H-thioxanthen-4-
yl)-6-morpholin-4-yl-pyran-4-one (13)
O,. ~ O
OH OH ,S\ /F
'~ F
\ F
a ~ ~ S ~ \ b
/ \
F O F O F
11
F /
O
S O \ O NJ
c / ~ ~ \ d
\ / \
O
O F
12 13
a: HzS04, Thiosalicylic acid; b: Tf20, pyridine; c: bis(pinacolato)diboron,
PdCl2dppf, dppf,
dioxane,100°C; d: chloropyranone, Pd(PPh3)4, dioxane, 90°C
5 (a) 1-Fluoro-4-hydroxy-thioxanthen-9-one (10)
Thiosalicylic acid (46.26 g, 0.3 mol) and 4-fluorophenol
(56.05 g, 0.5 mol) were dissolved in cons. HZSO9 (750 ml) and
the mixture was stirred under nitrogen for 24 hours. The
reaction mixture was then poured onto ice (1.5 L) and the
10 yellow precipitate was filtered and washed with water (300
ml). The precipitate was dried at 50°C for 24 hours and was
used without further purification (31.4 g, 42.50). m/z (LC-MS,
ESP) : 247 (M+ +1 ) .
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(b) I-fluoro-9-oxo-thioxanthen-4-yl trifluoromethane sulfonate
(11)
1-Fluoro-4-hydroxy-thioxanthen-9-one (4.92 g, 20 mmol) was
dissolved in dry pyridine (100 ml) and cooled to 0°C under a
5 nitrogen atmosphere. Triflic anhydride (3.66 ml, 22.3 mmol)
was added drop wise to the stirred solution over 5 minutes.
The reaction was left overnight and was then poured onto water
(300 ml) and the precipitate formed was filtered. The solid
was purified through a plug of silica (ethyl acetate: hexane;
10 1:9) to give the title compound as a white fluffy solid (1.72
g, 22.4 0) . m/z (LC-MS, ESP) : 379 (M+ +1) .
(c) I-Fluoro-4-(4,4,5,5-tetramethyl-(1,3,2)dioxaborolan-2-yl)-
thioxanthen-9-one (12)
15 1-fluoro-9-oxo-thioxanthen-4-yl trifluoromethane sulfonate
(11)' (378 mg, 1 mmol), bis(pinacolato)diboron (305 mg, 1.2
mmol), and ground potassium acetate (294mg, 3mmo1) were
suspended in dioxane (5 ml) and degassed (sonication for 5
minutes then saturated with NZ). PdClzdppf (40 mg, 0.050 mmol)
20 and dppf (27.7 mg, 0.05 mmol) was then added and the reaction
mixture was then heated at 100°C for 24hrs under a vigorous
stirring and a Nz atmosphere. The solvent was removed in vacuo
and the residue was purified by column chromatography (silica;
ethyl acetate: ethanol; 9:1) to give an oil which was used
25 without further purification (116 mg, 320). m/z (LC-MS, ESP):
357 (M+ +1).
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(d) 2-(1-Fluoro-9-oxo-thioxanthen-4-yl)-6-morpholin-4-y1-
pyran-4-one (13)
2-Chloro-6-morpholin-4-yl-pyran-4-one (3) (100 mg, 0.46 mmol),
1-Fluoro-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-
thioxanthen-9-one (12) (110 mg, 0.31 mmol), and ground
potassium carbonate (63mg, 0.62 mmol) were suspended in
dioxane (5 ml) and degassed (sonication for 5 minutes then
saturated with N2). Pd(PPh3)q (18 mg, 0.016 mmol) was then
added and the reaction mixture was then heated at 90°C for 24
hours under a vigorous stirring and a NZ atmosphere. The
solvent were removed in vacuo and the residue was then
suspended in water (50 ml) and extracted with ethyl acetate (3
x 50 ml). The organics were combined, washed with saturated
brine and dried over sodium sulphate. The solvent was removed
in vacuo and the residue was purified by column chromatography
(silica; ethyl acetate: ethanol; 9:1) to give the title
compound as a white solid (5 mg, 40). m/z (LC-MS, ESP): 410
(M+ +1 ) .
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Example 7: Synthesis of 2-(1-Fluoro-9H-thioxanthen-4-yl)-6-
morpholin-4-yl-pyran-4-one
(17)
O O
S~~' F
OH OH O \_F
S F
S I ~ a / ( S I W b / I I
w / ~ w / ~ /
O ~F F F
14 15
O~ ,O F / ( ~O
B
O NJ
/ I I \ d \ S I I
W /
/ O
16 F 17
a: BH3 THF; b: TfzO, pyridine; c: bis(pinacolato)diboron, PdCl2dppf, dppf,
dioxane,100°C; d: chloropyranone, Pd(PPh3)4, dioxane, 90°C
(a) 1-Fluoro-4-hydroxy-thioxanthen-9-one (14)
1-Fluoro-4-hydroxy-thioxanthen-9-one (4.93 g, 20 mmol) was
dissolved in THF (50 ml) and cooled down to 0°C under a N2
atmosphere. Borane-tetrahydrofuran complex (1M, 60 ml, 60
mmol) was added drop wise to the stirred solution over 10
minutes. The reaction was left to react overnight and was then
quenched with acetone (100 ml). The mixture was evaporated to
dryness and the residue was taken into water (200 ml). The
product was extracted in ethyl acetate (3 x 100 ml) and the
organics were combined, dried over sodium sulphate and
evaporated in vacuo. The residue was purified by column
chromatography (silica, hexane:ethyl acetate, 9:1) to give a
white solid which is readily oxidised by air (2.19 g, 47o). 1H-
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NMR (300MHz, DMSO-d6): 8H = 3.86 (2H, s); 6.73 (1H, m); 6.95
(1H, m) ; 7.24 (2H, m) 7.47 (2H, m) ; 10.07 (1H, s) .
(b) 1-fluoro-9H-thioxanthen-4-yl trifluoromethane sulfonate
(15)
1-Fluoro-4-hydroxy-thioxanthen-9-one (1.66 g, 7.15 mmol) was
dissolved in dry pyridine (35 ml) and cooled to 0°C under a
nitrogen atmosphere. Triflic anhydride (2.22 g, 7.87 mmol) was
added dropwise to the stirred solution over 5 minutes. The
reaction was left to react for 4 hours at room temperature and
was then pour onto water (350 ml). The milky solution was
extracted with DCM (3 x 200 ml), the organics were combined
and dried over magnesium sulphate. The solvent was removed in
vacuo and the solid obtained was purified through a plug of
silica (ethyl acetate: hexane; 3:97) to give the title compound
as a white fluffy solid (2.55 g, 98o). 1H-NMR (300MHz, DMSO-
d6): 8H = 3.86 (2H, s); 7.3-7.6 (6H, m)
(c) I-Fluoro-~-(4,4,5,5-tetramethyl-(1,3,2Jdioxaborolan-2-y1)-
9H-thioxanthe (16)
1-fluoro-9H-thioxanthen-4-yl trifluoromethane sulfonate (1 g,
2.75 mmol), bis(pinacolato)diboron (840 mg, 3.30 mmol), and
ground potassium acetate (809 mg, 8.25 mmol) were suspended in
dioxane (7 ml) and degassed (sonication for 5 minutes then
saturated with NZ). PdCl2dppf (0.112 mg, 0.138 mmol) and dppf
(77 mg, 0.138 mmol) was then added and the reaction mixture
was then heated at 100°C for 24 hours under a vigorous
stirring and a NZ atmosphere. The solvent were removed in
vaccuo and the residue was purified by column chromatography
(silica; ethyl acetate:ethanol; 9:1) to give an oil which was
used without further purification (460 mg, 49%).
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(d) 2- (1-Fluoro-9H-thioxanthen-~-yl) -6-morpholin-~-yl-pyran-4-
one (17)
2-Chloro-6-morpholin-4-yl-pyran-9-one (3)(252 mg, 1.17 mmol),
1-Fluoro-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-9H
thioxanthe (400 mg, 1.17 mmol), and ground potassium carbonate
(239 mg, 2.34 mmol) were suspended in dioxane (7 ml) and
degassed (sonication for 5 minutes then saturated with NZ).
Pd(PPh3)q (67 mg, 0.059 mmol) was then added and the reaction
mixture was then heated at 90°C for 24hrs under a vigorous
stirring and a Nz atmosphere. The solvent were removed in
vaccuo and the residue was purified by column chromatography
(silica; ethyl acetate: ethanol; 9:1) to give an off white
solid which was triturated in ether and gave the title
compound as a white solid (72.3 mg, 160). 1H-NMR (300MHz, DMSO-
d6) : 8H = 3.41 (4H, t) ; 3.71 (4H, t) 5.50 (1H, d) ; 6.21 (1H,
d); 7.25-7.35 (3H, m); 7.52-7.62 (3H, m). m/z (LC-MS, ESP):
396 (M+ +1).
Example 8: Synthesis of 2-Thianthren-1-yl-6-morpholin-4-yl-
pyran-4-thione (18)
O ~O ( \ ~O
S / S / O NJ
Lawesson's reagent
\ Toluene, reflux \
O I / S
(18)
2-Thianthren-1-yl-6-morpholin-4-yl-pyran-4-one (9)(140 mg,
0.354 mmol) was dissolved in toluene (5 ml). Lawesson's
reagent (215 mg, 0.53 mmol) was added to the solution and the
mixture was refluxed overnight under nitrogen with stirring.
The toluene was evaporated off in vacuo and the residue was
purified via column chromatography (silica, dichloromethane)
to give the desired compound (18) as dark orange solid (27 mg,
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18 0) .1H-NMR (300MHz, DMSO-d6) : 8H = 3.56 (9H, t, J 5Hz) : 3. 73
(4H, t, J 5Hz); 7.83 (1H, d, J 2Hz); 7.76 (1H, d, J 2Hz);
7.30-7.80 (7H, m). m/z (LC-MS, ESP):412 (M+ +1).
5 Example 9: 2-(7-Amino-9H-thioxanthen-4-yl)-6-morpholin-4-yl-
pyran-4-one N-amide derivatives
/ O~
\ Br S OMe
\ ~ \ S
OzN / OH ~ ~ / OH /
OzN ~ ~ O N U ~ a
O z
O O
OH
\ S I \ I \ S I \ I \ S
/ /
OzN HzN HZN
O~ ,O
OH OTf B
\ S ~ \ ~ ~ \ S ( \ ~ \ S ~ \
BocNH / / BocNH / / BocNH / /
~O n0
N J
BocNH Hz
(19) (20)
2-(2-Methoxy-phenylsulfanyl)-5-nitro-benzoic acid
10 2-Methoxythiophenol (9.9m1, 81.29mmo1) was added to a solution
of KOH (18.298, 325.18mmo1) in water (80m1) degassed for 15
minutes. 2-Bromo-5-nitrobenzoic acid (20.08, 81.29mmol) and
copper bronze (258mg, 4.06mmol) were added to the reaction
mixture, which was refluxed overnight. The reaction was
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stopped and the mixture was filtered through a celite pad and
washed with 2M NaOH then water (50m1). The filtrate was
acidified (pH 1) with concentrated HCl. The precipitate formed
was filtered and dried overnight in a vacuum oven (50°C) to
give the crude title compound (26.0 g) as a pale yellow solid.
The product was used without further purification.
5-Methoxy-2-nitro-thioxanthene-9-one
2-(2-Methoxy-phenylsulfanyl)-5-nitro-benzoic acid (13.008,
42.58mmo1) was suspended in methanesulphonic acid (100m1) and
heated at 100°C. The crude mixture was slowly poured onto ice
with vigorous stirring then neutralized with conc. ammonia
solution. The precipitate was filtered and washed with water.
The yellow/ lime colored solid was dried under vacuum at 50°C
to give the crude title compound which was used without any
further purification (128, 980). m/z (LC-MS, ESP), RT=4.89
min, (M++1 ) = 288 .
5-Methoxy-2-vitro 9H-thioxanthene
To a cooled (0°C) suspension of 5-methoxy-2-nitro-
thioxanthene-9-one (24.468, 85.13mmo1) in anhydrous
tetrahydrofuran (40m1) under nitrogen atmosphere, was added
drop wise borane-THF complex (170m1, 1. OM in THF). The mixture
was allowed to warm to room temperature with stirring
overnight. The reaction mixture was cooled (0°C) and the
excess borane was quenched with acetone. The solvent was
evaporated in vacuo. The residue was purified by flash
chromatography (1:1, dichloromethane/hexane) to give the title
compound (11.598, 500) as a bright yellow amorphous solid.
1HNMR (300MHz, DMSO-d6) : 8H= 3.86 (3H, s) , 4.03 (2H, s) , 7.00
(2H, dd), 7.28 (1H, t), 7.73 (1H, d), 8.05 (1H, dd), 8.28 (1H,
d) .
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5-Methoxy-9H-thioxanthen-2-ylamine
5-Methoxy-2-nitro 9H-thioxanthene (11.598, 42.40mmo1) was
suspended in ethyl acetate (250m1). SnC12.2H20 (47.848,
212mmo1) was added and the clear yellow solution was stirred
at 50°C overnight. The reaction was the quenched with NaOH
(2M) and then extracted with ethyl acetate (3x300m1). The
organics were washed with saturated brine (100m1), dried over
magnesium sulphate and the solvents were removed in vacuo to
give the title compound (10.328, 1000) as viscous yellow oil.
The oil was used without further purification. . 1HNMR (300MHz,
DMSO-d6) : 8H= 3.83 (3H, s) , 3. 67 (2H, s) , 5. 14 (2H, bs) , 6.43
(1H, dd) , 6. 61 (1H, d) , 6.89 (1H, d) , 6. 99 (1H, d) , 7.06 (1H,
d), 7.18 (1H, t). m/z (LC-MS, ESP), RT=3.88 min, (M+ +1)= 244.
7-Amino-9H-thixanthen-~-of
5-Methoxy-9H-thioxanthen-2-ylamine (10.328, 41.09mmo1) and
pyridine hydrochloride (49.08, 424mmo1) were heated at 200°C
under nitrogen atmosphere for 5 hours. The black reaction
mixture was allowed to cool down to room temperature and water
(50m1) was then added. The mixture was neutralized with NaOH
(2M) to pH 7 then extracted with dichloromethane (4x100m1).
The organics were washed with saturated brine, dried over
MgS09) and concentrated in vacuo to give a black oil. This oil
was purified by flash chromatography (dichloromethane) to give
the title compound (7.788, 800) as dark brown oil which was
used without further purification. 1HNMR (300MHz, DMSO-d6): 8H=
3. 61 (2H, s) , 5.08 (2H, bs) , 6. 42 (1H, dd) , 6. 58 (1H, d) , 6. 69
(1H, d) , 6. 81 (1H, d) 6. 95-7.06 (2H, m) , 9.88 (1H, bs) ; m/z
(LC-MS, ESP), RT= 3.23 min, (M+ +1)= 230.
(5-Hydroxy-9H-thioxanthen-2-y1)-carbamic acid tert-butyl ester
To a solution of 7-amino-9H-thixanthen-4-of (7.77 g, 81.32
mmol) in THF (14 ml) was added dropwise di-tert-butyl
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dicarbonate (17.74 mg, 0.49 mmol) in THF (4 ml). The reaction
was stirred at room temperature under nitrogen atmosphere.
Upon completion of the reaction the solvent was evaporated.
The residue was taken up in methanol (50m1), and sodium
hydroxide (4.06g, 101.16mmo1) was added. The dark brown
mixture was refluxed for 20 minutes. The solvent was
evaporated in vacuo and the oil was taken up in water,
extracted with ethyl acetate, dried over MgSOq and evaporated
in vacuo to give the crude product. The dark brown oil was
purified by flash chromatography (dichloromethane) to give the
title compound (4.2g, 380), as a cream coloured amorphous
solid. 1HNMR (300MHz, DMSO-d6) : 8H= 3.74 (2H, s) , 6.74 (1H, d) ,
6.87 (1H, d), 7.04 (1H, t), 7.23-7.33 (2H, m), 7.57 (1H, bs),
10.03 (1H, bs).
(5-Trifluoromethanesulfonyl-9H-thioxanthen-2-yl)-carbamic acid
tert-butyl ester
To a cooled (0°C) golden colored solution of (5-hydroxy-9H-
thioxanthen-2-yl)-carbamic acid tert-butyl ester (4.Og,
12.14mmo1) in anhydrous pyridine (8m1) under nitrogen
atmosphere was added trifluoromethanesulphonic anhydride
(2.36m1, 13.35mmo1) drop wise. The solution turned deep orange
upon addition of trifluoromethanesulphonic anhydride. The
reaction was allowed to warm to room temperature. After 10
minutes of stirring at this temperature the solution was
poured into water (20m1). The product was extracted with
ethyl acetate. The organics were washed with saturated brine,
dried over MgS04 and concentrated in vacuo to give the title
compound (5.6g, 1000) as a dark orange solid.
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[5-(9,4,5,5-Tetramethyl-[1,3,2Jdioxaborolan-2-y1)-9H-
thioxanthen-2-y1)-carbamic acid tert-butyl ester
(5-Trifluoromethanesulfonyl-9H-thioxanthen-2-yl)-carbamic acid
tert-butyl ester (3.318, 7.17mmo1), bis(pinacolato)diboron
(2.18g, 8.6mmo1) and potassium acetate (2.118, 21.5mmo1) in
1,4-dioxane (20m1) was degassed for 15 minutes. To the yellow
suspension was then added PdCl2(dppf) (293mg, 0.36mmo1) and
dppf (199mg, 0.36mmo1). The dark red mixture was heated to
90°C under a Nz atmosphere for 48 hours. The crude mixture was
purified by flash chromatography (dichloromethane) to give
viscous brown oil (3.15g), which was used without any further
purification.
[5-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-y1)-9H-thioxanthen-2
yl]-carbamic acid tert-butyl ester (19)
[5-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-9H-
thioxanthen-2-yl]-carbamic acid tert-butyl ester (1.02g,
2.32mmo1), 2-chloro-6-morpholin-4-yl-pyran-4-one (3) (0.608,
2.78mmo1) and KZC03 (0.648, 4.64mmo1) were dissolved in dry
1,4-dioxane (ml). The mixture was degassed for 15 minutes and
Pd(PPh3)4 (0.13g, 0.12mo1) was then added The dark brown
mixture was heated to 90°C under an atmosphere of NZ for 24
hour. The reaction mixture was concentrated in vacuo and water
(50m1) was added. The brown solid was filtered and washed with
water (1.218, 880). m/z (LC-MS, ESP), RT = 4.6 minutes, (M++1)=
493.
2-(7-Amino-9H-thioxanthen-4-y1)-6-morpholin-4-y1-pyran-4-one
(20)
To a solution of [5-(6-Morpholin-9-yl-4-oxo-4H-pyran-2-yl)-9H-
thioxanthen-2-yl]-carbamic acid tert-butyl ester (19)(1.08 g,
2.19 mmol) in dichloromethane (10 ml) was added
trifluoroacetic acid (2 ml) and left under stirring at room
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temperature overnight. The solvent was dried in vacuo
revealing a viscous dark brown liquid. Saturated sodium
bicarbonate solution (20 ml) was added to the residue, which
was left to stir for 20 mins. The brown precipitate was
5 filtered, washing with water and left to dry in the vacuum
oven overnight (0.77g, 900). 1HNMR (300MHz, DMSO-d6): 8H= 3.40
(4H, t) , 3.70 (4H, t) , 3.77 (2H, s) , 5.23 (2H, bs) , 5.50 (1H,
d) , 6.17 (1H, d) , 6:44 (1H, dd) , 6. 65 (1H, d) , 7.09 (1H, d) ,
7.35 (1H, t), 7.47-7.59 (2H, m); m/z (LC-MS, ESP), RT= 3.51
10 minutes, (M++1 ) = 392 .
2-(7-Amino-9H-thioxanthen-4-y1)-6-morpholin-4-y1-pyran-4-one
N-amide derivatives
15 (a) To a small test tube was added 2-(7-amino-9H-thioxanthen-
4-yl)-6-morpholin-4-yl-pyran-4-one (20)(20mg, 0.05mmol), dry
dimethylacetamide (0.5m1), triethylamine (O.Olml, 0.08mmo1)
and the desired acid chloride (0.08mmol) with stirring
overnight. The reaction was purified by preparative HPLC to
20 give the desired products, which are shown below:
O
R~N
H
/ I ~o
O NJ
/ O
Compound R Purity Retention M++1
Time (Mins)
21 ~ ~ 90 3.46 435
~ ~ o
22 ~~. 90 3 . 62 4 65
23 I 90 3.58 493
o~y
I I
0
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24 ~ 95 3.82 961
25 ~°~. 95 3.66 521
0
26 N 90 3.53 498
~I,
27 ~ 90 4.16 487
28 N~ 90 3.43 498
29 ~ °~ 95 9.94 527
30 ~ 90 4.1 503
31 95 4.03 517
s
32 ~ 95 3.99 475
33 0 90 9.13 516
N~ I
34 ~ 90 3.64 479
~o
35 "° 90 4.12 517
N~ I
N =
36 ~ 90 3.43 546
N
37 ~ °~ 90 3.91 555
w I = o
38 ~ o' 90 4.16 587
0
I
~o ~
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39 °II 90 3.59 507
~o~*
40 ~ ' 90 3.5 493
o~
41 w 90 4.1 569
I
0
~o
42 -~ 85 4.31 515
43 0 ~ 95 9.16 539
I
44 °1 95 4.46 573
0
I
F \
45 °'N 95 4.02 502
I ,
46 I ~ S~ 95 4.72 586
N
47 Nr ~ 95 3.67 488
o
48 ~~ 95 3.42 493
HO
49 H°~' 95 3.38 505
0
50 o FF 90 3.48 565
HO~
F F
(b) To a small test tube was added 2-(7-amino-9H-thioxanthen-
4-yl)-6-morpholin-4-yl-pyran-4-one (20)(20mg, 0.05mmol), dry
dimethylacetamide (0.5m1), triethylamine (8p1, 0.06mmo1) and
chloroacetyl chloride (4u1, 0.06mmo1) with stirring overnight.
The appropriate amine or thiol (20mg or 201) was then added
and left to stir at room temperature overnight. The reaction
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was purified by preparative HPLC to give the desired products,
which are shown below:
0
R~N
H
i
O NJ
0
Compound R Purity Retention Time M++1
(Mins)
51 N~. 90 3.07 518
52 ~N~= 95 2.98 519
HN J
53 ~N~= 95 2.98 533
/NJ
54 H~N~* 95 2.98 538
OH
55 ~~N~* 95 3.23 566
,o
56 H~N~. 95 2.93 494
H
57 HzN~N~* 95 2.74 493
H
58 N~* 95 3.06 504
59 ~N~. 90 2.99 547
NJ
60 ~N~. 95 2.93 533
N
H
61 ~* 95 3.82 532
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62 ~N~N~= 95 2.77 577
0
J "
63 w N~. 95 3.18 570
I
H
\O
64 /r~ 95 3.4 534
~
N
~
g
.
65 ~ 95 3.1 506
\iN~N~.
H
66 HzN~. 95 2.96 450
67 0~ 95 2.97 563
~N~N~.
H
68 N~. 95 3.16 534
~
H
(c) To a small test tube was added 2-(7-amino-9H-thioxanthen-
4-yl)-6-morpholin-4-yl-pyran-4-one (20) (20mg, 0.05mmol), dry
dimethylacetamide (0.5m1), triethylamine (8u1, 0.06mmo1) and
3-bromopropionyl chloride (5~1, 0.05mmol) with stirring
overnight. The appropriate amine or thiol (20mg or 20u1,
hydrochloride salts were freed by addition of triethylamine)
was then added and left to stir at room temperature overnight.
The reaction was purified by preparative HPLC to give the
desired products, which are shown below:
O
R~N
H
~o
O NJ
S
O
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Compound R Purity Retention Time M++1
(Mins)
69 90 3.17 532
N~;
70 HN~ 90 2.88 533
~N~~
71 ~N~ 95 2.98 547
~N~,
72 95 2.95 552
HO~N~
73 ~0 95 3.19 580
wO~N~.
74 ~N~ 95 2.95 508
HO
75 ~,",,~ 90 2.76 507
HZN
,
76 95 3.08 518
N~
77 ~ 90 2.81 561
N
~N~Y
7g ,"~ 95 2.83 547
~N~~
79 ~ 90 3.2 546
N~
80 0~ 95 2.84 591
H
~N~N~~
81 I 95 3.3 584
0
H
I N
~.
82 <N~S~. 95 3 . 43 598
N-NH
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83 ~ 90 3.06 520
~N~
84 "=N~. 90 2.98 464
85 0~ 95 2.89 577
H
~N~N~
86 /'o 95 3.14 548
H
~N~
Example 10: 2-(4-Hydroxy-9H-thioxanthen-lyl)-6-morpholin-4-
yl-pyran-4-one ether derivatives
OH OH
SH I \ S I \ I \ S
OH / / ~ / /
O O Br Br
OBOC OBOC HO / O
\ S \ \ S \
S \ ( O NJ
/ / / /
Br O~B.O I / O
(87)
1-Bromo-~-hydroxy-thioxanthen-9-one
Thiosalicylic acid (20.08, 129.71mmo1) and 4-bromophenol
(35.98, 207.53mmo1) were suspended in cons. HZSO9 (200m1) and
stirred for 48 hours. The red solution was slowly poured onto
ice (500m1) with vigorous stirring. The resulting yellow
precipitate was filtered, and dried in a vacuum oven (50°C) to
give the title compound (24.238, 610) as a yellow amorphous
solid. m/z (LC-MS, ESP), RT=4.39 min, (M--1)= 305-307.
1-Bromo-9H-thioxanthen-Q-o1
To a cooled (0°C) suspension of 1-bromo-4-hydroxy-thioxanthen-
9-one (24.238, 78.88mmo1) in anhydrous tetrahydrofuran (40m1)
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under nitrogen atmosphere, was added dropwise borane-THF
complex (237m1, 1M in THF). The cloudy mixture was allowed to
warm to room temperature and was left stirring overnight. The
suspension dissolved gradually as the reaction progressed
giving a yellow solution. The reaction mixture was cooled (0°C)
and the excess borane was quenched with acetone. The yellow
solution was evaporated in vacuo. The resulting oil was
purified by flash chromatography (4:1, hexane / ethyl acetate)
to give the title compound (11.508, 500). m/z (LC-MS, ESP),
RT=4.84 min, (M- -1)= 291-293.
Carbonic acid tert-butyl ester 1-(4,9,5,5-tetramethyl-
(1,3,2Jdioxaborolan-2-y1)-9H- thioxanthen-4-y1 ester
To a stirred solution of 1-bromo-9H-thioxanthen-4-of (11.508,
39.22mmo1) in pyridine (7ml) was added triethylamine (8.15m1,
58.83mmo1). To the solution was added dropwise di-tert-butyl
dicarbonate (9.418, 3.14mmo1) in pyridine (4m1). After 1 hour
of stirring the crude reaction mixture was poured into water
(100m1) and extracted with dichloromethane (3x100m1). The
organics were washed with sat. brine (50m1), dried over MgSOq
and the solvent was evaporated in vacuo to give the title
compound (10.408, 670) as a clear viscous oil. 1HNMR (300MHz,
DMSO-d6): ~H= 1.53 (9H, s), 4.09 (2H, s), 7.15-7.65 (6H, m).
Carbonic acid tert-butyl ester 1-(9,4,5,5-tetramethyl
(1,3,2]dioxaborolan-2-yl)-9H-thioxanthen-4-yl ester
Carbonic acid tert-butyl ester 1-(4,4,5,5-tetramethyl-
[1,3,2]dioxaborolan-2-yl)-9H-thioxanthen-4-yl ester (5.008,
12.71mmo1), anhydrous potassium acetate (3.798, 38.13mmo1),
1,1'-bis(diphenylphosphino)ferrocene (352mg, 0.64mmo1) and
bis(pinacolato)diboron (3.878, 15.25mmo1) were suspended in
anhydrous dioxane (8m1) under nitrogen atmosphere. The mixture
was degassed for 10 minutes and dichloro[1,1'-
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bis(diphenylphosphino)ferrocene]palladium(II) dichloromethane
adduct (514mg, 0.64mmo1) was added to the mixture. The
reaction was heated at 90°C under nitrogen atmosphere for 24
hours. The crude reaction mixture was purified by flash
chromatography (dichloromethane), to give the title compound
(3.02g) as a crude brown oil which was used without further
purification.
2-(~-Hydroxy-9H-thioxanthen-lyl)-6-morpholin-9-yl-pyran-~-one
(87)
Carbonic acid tert-butyl ester 1-(4,4,5,5-tetramethyl-
[1,3,2]dioxaborolan-2-yl)-9H-thioxanthen-4-yl ester (3.OOg,
6.81mmo1), 2-chloro-6-morpholin-4-yl-pyran-4-one (1.228,
5 . 67mmo1 ) and potassium carbonate ( 2 . 07g, 14 . 98mmo1 ) were
suspended in anhydrous dioxane (6ml) under nitrogen
atmosphere. The solution was degassed for 15 minutes. To the
solution tetrakis(triphenylphosphino) palladium (291mg, 50
eq.) was added. The mixture was degassed for a further 5
minutes. The reaction was heated at 90°C under nitrogen
atmosphere for 24 hours. The solvent was evaporated in vacuo
and the crude mixture was purified by column chromatography
(9:1, ethyl acetate/ethanol), to yield the title compound
(421mg, 160) as a light yellow amorphous solid. 1H NMR (300MHz,
DMSO-d6) : 8H= 3.33 (4H, t) , 3. 67 (4H, t) , 3.88 (2H, s) , 5. 45
(1H, d), 6.05 (1H, d), 6.87 (1H, d), 7.24-7.65 (5H, m), 10.62
(1H, bs) ; m/z (LC-MS, ESP) , RT=3. 96 min, (M++1)= 394.
2-(4-Hydroxy-9H-thioxanthen-ly1)-6-morpholin-4-yl-pyran-9-one
ether derivatives
(a) To a mixture of 2-(9-hydroxy-9H-thioxanthen-lyl)-6-
morpholin-4-yl-pyran-4-one (87)(20mg, 0.05mmo1) and potassium
carbonate (l6mg, 0.11mmo1) in N,N-dimethylformamide (0.5m1)
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was added dibromoethane (22u1, 0.25mmol). After 4 hours the
appropriate amine or thiol (0.254mmo1, 5eq) was added to the
solution, and the compounds isolated are shown below:
R / ~O
o NJ
/ 0
Compound R Purity Retention Time M++1
(Mins)
88 ~ 95 3.26 505
N
O~
89 N' 95 3.00 506
CN
O~
90 I 95 3.13 520
CND
,
91 HO~ 'OH 95 3.03 525
Jr
N
O
92 ~~ ~~ 95 3.3 553
N
O~
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93 ~o" g5 3.02 481
HN
O~
g9 NHZ 95 2.76 480
HN
O~
°" 85 3.03 511
HO
HN
0~
96 ~ 90 3.15 491
N
O~
g7 / 90 2.88 534
N
O
gg N 90 2.83 520
c~
o~
95 3.28 519
N
O~
100 ~N~ 95 3.08 465
o~
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101 0~ 95 3.38 557
I
i
HN
O~
102 ~~ 90 3.7 521
N
,
~
N
S
O
(b) 2-(4-hydroxy-9H-thioxanthen-1yl)-6-morpholin-4-yl-pyran-9-
one (87)(20 mg, 0.0508 mmol), potassium carbonate (44mg, 0.315
mmol) and N,N-dimethylformamide (0.5m1) was added to 2, 3 or
4-picolyl chloride hydrochloride (0.25mmo1), respectively.
The reactions were stirred at room temperature for 2 hours.
The crude reaction mixtures were submitted for purification by
preparative HPLC without any further workup, and the compounds
produced are shown below:
R
I
~O
N J
Compound R Purity Retention M++1
Time (Mins
)
103 i N 95 4.07 485
I
o,
104 NI 90 3.52 485
o~
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105 N~ I 90 3.37 485
o~
Example 11: N-Acyl 2-(1-Amino-9H-thioxanthen-4-yl)-6-
morpholin-4-yl-pyran-4-one derivatives
OH OBn
SH I ~ S I ~ ~ I ~ S
/ OH / / / /
O O F O F
OBn OBn O
S I W ~ W S ~ W ---r I \ S I W
/ /
O NPMB NPMB N
OH OTf O.B~O
/ / / / / /
NBOC NBOC NBOC
BOCHN / I O ~O H2N / I ~O
O NJ
~~ ~
/J o ~ ;~ 'o
(106)
1-Fluoro-9-hydroxy-thioxanthen-9-one
To a solution of 2-thiosalicylic acid (39.328, 255 mmol) in
concentrated sulfuric acid (700 ml) was added 4-fluorophenol
(32.0 g, 280 mmol). The red solution was then stirred at room
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temperature for 18 hours. Upon completion, the mixture was
poured directly onto 4 litres of crushed ice and the resulting
red solid was filtered off, and then suspended in water (1
Land treated with ammonia solution until pH 6 attained
whereupon the precipitate was re-filtered to give the title
compound as an orange solid (44.48 g, 70.80) m/z (LC-MS, ESP):
247 [M+HJ+, R/T = 3.99 mins
4-Benzyloxy-1-fluoro-thioxanthen-9-one
KZC03 (21.0 g, 150 mmol) was added to a stirred suspension of
1-Fluoro-4-hydroxy-thioxanthen-9-one (18.97 g, 75.0 mmol) in
methanol (100 mL) followed by benzylbromide (16 mL, 75.0 mmol)
which was added in slow stream via syringe. The resulting
mixture was then heated to reflux for 90 minutes and then
cooled to room temperature before it was poured onto cruched
ice (0.5 L). The resulting precipitate was filtered off and
dried (P205) to give the title compound as a yellow solid (16.7
g, 66.10) m/z (LC-MS, ESP) : 337 [M+H]+, R/T = 5.22 mins
~-Benzyloxy-1-(~-methoxy-benzylamino)-thioxanthen-9-one
To a solution of 4-methoxybenzyl amine (1.63 g, 11.89 mmol) in
dry pyridine (10 ml) was added 4-Benzyloxy-1-fluoro-
thioxanthen-9-one (1 g, 2.97 mmol) in a single portion. The
mixture was then heated to reflux (140 °C) for 18 hrs. The
resulting hot orange suspension was allowed to cool to room
temperature before being poured onto 100 ml of crushed ice.
The precipitate was filtered off and washed with copious
amounts of water to give the title compound as a red/orange
solid (1.35 g, 89.6%). m/z (LC-MS, ESP): 454 [M +HJ+ R/T = 6.09
mins.
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(4-Benzyloxy-9H-thioxanthen-1-y1)-(9-methoxy-benzyl)-amine
To a cooled (0°C) suspension of 4-benzyloxy-1-(4-methoxy-
benzylamino)-thioxanthen-9-one (8.16 g, 18.00 mmol) in dry THF
(150 ml) was added Borane-THF complex (90 mmol, 90 ml 1M in
THF) in a dropwise fashion. The reaction was allowed to
slowly warm to room temperature.and stirred for a further 16
hours to give a homogeneous yellow solution. To mixture was
then cooled (0°C) and diluted slowly with acetone (150 ml) and
then stirred for 60 minutes at room temperature. The solvent
was removed in vacuo to give a crude residue that was diluted
in CHzCl2 (100 ml) and the washed with a saturated solution of
NaHC03 (100 ml), dried using MgS04, filtered and concentrated
in vacuo to give the title compound as a mild amber oil (7.90
g, 99.80) m/z (LC-MS, ESP): 438 [M+HJ+, R/T = 5.01 mins.
1-Amino-9H-thioxanthen-4-of
(4-Benzyloxy-9H-thioxanthen-1-yl)-(4-methoxy-benzyl)-amine
(14.51 g, 33.00 mmol) was mixed thoroughly with solid pyridine
hydrochloride (190 g, 165.00 mmol) before being heated to
150°C and stirred at this temperature for a further 12 hours.
Upon completion the reaction was cooled slightly before being
poured into an beaker of ice/water. The brown precipitate was
removed by filtration and the filtrate adjusted to pH 11 with
NH30H solution before being extracted with CHZCIz (3x100 ml).
The combined organic phases were then washed with water (1x100
ml) and brine (1x100 ml) then dried using MgS04, filtered and
concentrated in vacuo to give the title compound as a thick
brown oil (7.50 g, 99.10) m/z (LC-MS, ESP): 229 [M+H]+, R/T =
4.15 mins.
(4-Hydroxy-9H-thioxanthen-1-yl)-carbamic acid tert-butyl ester
To a solution of 1-amino-9H-thioxanthen-4-of (7.57 g, 33.00
mmol) in dry THF (50 ml) was added di-tertiary butyl
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dicarbonate (20 g, 91.64 mmol) in a single portion. The
reaction was stirred at room temperature for 4 hours before
the addition of methanol (50 mL) and solid NaOH (10 g, 250
mmol). The resulting slurry was stirred at room temperature
5 for 1 hr before the addition of H20 (250 ml) and EtOAc (250
ml). The organic extract was removed and the remaining
aqueous extracted further with EtOAc (2x50 ml). The combined
organics were then dried using MgS04, filtered and
concentrated in vacuo to give the title compound a dark amber
10 oil (10.87 g, 92 0) m/z (LC-MS, ESP) : 328 [M-H]-, R/T = 4.73
mins
Trifluoro-methanesulfonic acid 1-tert-butoxycarbonylamino-9H-
thioxanthen -4-yl ester
15 To a cooled (0°C) solution of (9-Hydroxy-9H-thioxanthen-1-yl)-
carbamic acid tent-butyl ester (10.05 g, 30.50 mmol) in dry
pyridine (70 ml) was added trifluoromethanesulphonic anhydride
(8 ml, 48.77 mmol) in a slow stream via syringe over 10 mins.
The brown mixture was stirred at 0°C for a further 30 mins
20 before the addition of water in a dropwise fashion. The
mixture was extracted with EtOAc (3x100 mL), the organic
extracts combined, dried using MgS04, filtered and
concentrated in vacuo to give a pale brown oil. Purification
of the crude residue was accomplished by flash chromatography
25 (Si02) using Hexanes:EtOAc (4:1) to give a mild amber oil that
was purified by flash chromatography (Si02) (Hexanes then 3:1 -
Hexanes:EtOAc) to give a mild amber oil (9.42 g, 67.0%) m/z
(LC-MS, ESP): 460 [M-H] , R/T = 5.52 mins.
30 (~-(9,~,5,5-Tetramethyl-(1,3,2Jdioxaborolan-2-yl)-9H
thioxanthen-1-ylJ-carbamic acid tent-butyl ester
To a solution of Trifluoro-methanesulfonic acid 1-tert-
butoxycarbonylamino-9H-thioxanthen-4-yl ester (3.05 g, 6.60
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mmol) in dry dioxane (10 ml) was added bis(pinacolato)diboron
(2.0 g, 7.92 mmol) and anhydrous potassium acetate (1.9 g,
19.80 mmol). The reaction was then degassed (sonication for
20 min then saturated with NZ) before the addition of
dichloro[l,1'-bis(diphenylphosphino)ferrocene] palladium(II)
dichloromethane adduct (0.26 g). The reaction mixture was
degassed for a further 20 minutes before a reflux condenser
was attached to the reaction vessel which was then heated to
100°C and stirred vigorously for 24 hours. The brown
reaction mixture was then poured onto a silica pad prepared in
hexanes and eluted with CHZCI2:Hexanes (1:1). The collected
eluent was concentrated in vacuo to give crude title compound
as a dark brown oil that was used without further purification
(2.90 g,).
(4-(6-Morpholin-4-yl-~-oxo-4H-pyran-2-yl)-9H-thioxanthen-1-
yl]-carbamic acid tert-butyl ester
[4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-9H-
thioxanthen-1-yl]-carbamic acid tert-butyl ester (2.90 g, 6.50
mmol) was introduced to a solution of 2-Chloro-6-morpholin-4-
yl-pyran-4-one (3)(1.4 g, 6.50 mmol) in anhydrous dioxane (6
mL). Powdered KZC03 (2.01 g, 14.50 mmol) was added and the
mixture degassed (sonication for 20 mins then saturated with
NZ). To the degassed solution was added Tetrakis
(triphenylphosphine) palladium (0.39 g) before it was degassed
for a further 20 minutes. A reflux condenser was attached to
the reaction vessel which was submerged into an oil bath
maintained at 100°C for 14 hours whereupon the golden mixture
was cooled and diluted with EtOAc (50 ml) and then washed with
water (20 ml) and saturated brine (20 ml). Organic extract
was dried using MgS04, filtered and concentrated in vacuo to
give the title compound as a light brown oil that was used
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without further purification. m/z (LC-MS, ESP): 493 [M+H]+, R/T
- 4.41 mins.
2-(I-Amino-9H-thioxanthen-~-yl)-6-morpholin-Q-yl-pyran-~-one
(106)
To a solution of [4-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-9H-
thioxanthen-1-yl]-carbamic acid tert-butyl ester (3.25 g) in
CHZC12 (25 ml) was added trifluoroacetic acid (5 ml). The
mixture was stirred at room temperature for 18 hrs wereupon it
was cooled (0°C) and quenched by dropwise addition of
saturated NaHC03 until the pH 9 was attained. The mixture was
then extracted using CHZCIz (3x20 mL), the combined organic
extracts were then dried (MgS04), filtered and concentrated in
vacuo to give a semi-crystalline solid that was applied onto a
thin silica pad and eluted with EtOAc (1000) going to
EtOAc:MeOH (9:1). The eluent was concentrated in vacuo to
give the title compound as a mild amber oil (1.46 g, 56.90
over three steps) m/z (LC-MS, ESP): 393 [M+H]+, R/T = 3.79 mins
N-Acyl 2-(1-Amino-9H-thioxanthen-~-y1)-6-morpholin-9-yl-pyran-
4-one derivatives
(a) To a stirred solution of 2-(1-Amino-9H-thioxanthen-4-yl)-
6-morpholin-4-yl-pyran-4-one (106)(39 mg, 0.1 mmol) in
anhydrous N,N-dimethylformamide (1 ml), N-
ethyldiisopropylamine (0.4 ml, 2.31 mmol) and 0-(7-
azabenzotriazol-1-yl)-N,N,N',N'-
tetramethyluroniumhexafluorophosphate (50 mg, 1.3 mmol) were
added. The appropriate carboxylic acid (0.1 mmol) was then
added and the mixture stirred at room temperature overnight.
The compound was then purified by preparative HPLC to give the
desired compounds, which are shown below:
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R\ / O
~O
NJ
Compound R Purity M++1
107 ~ 0 95 487
108 ~ 95 546
N
.
109 °~ 95 555
0
( .
110 95 580
~N
S
111 ~"'S 95 519
N
112 SN~ 95 555
N ~ .
113 Nc ~ 95 522
214 N ~ 95 4 98
I
115 /'° 85 541
0
I
116 ~ 95 479
~o
117 Nc N 99 537
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118 ~°~. 85 493
0
119 N~ 95 498
I
120 ~ 95 527
/o
[I
121 0~ 95 557
w
I
~o ~
122 ~ 95 544
N\ S
I
123 ~ 95 569
0 0
w
I
124 ~°~. 95 507
0
125 ~° 95 569
0
I
126 ~ 95 488
127 ~°~, 95 493
128 ~ 95 486
129 ~ 0 98 501
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(b) To a solution of 2-(1-amino-9H-thioxanthen-4-yl)-6-
morpholin-4-yl-pyran-4-one (106)(25 mg, 0.06 mmol) and
pyridine (0.5 mmol) in CH2Clz (1 mL) was added the appropriate
sulfonyl chloride (0.2 mmol) in a single portion. The
reaction was stirred at room temperature overnight. The
resulting reaction mixture was then purified by preparative
HPLC to give the desired compounds, which are shown below:
R
I
O=S=O
I
", ~O
N J
Compound R Purity M++1
130 I ~ 84 597
131 ° 97 57
HO
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Example 12: 2-Morpholin-4-yl-6-(11-oxo-10,11-dihydro-
dibenzo[b,f]thiepin-4-yl)-pyran-4-one
~O ~O ~O _
S S
/ ~ / ~ S
/
O
O O
O _ OTf O~B~O
S ~ ~ ~ S ~ ~ ~ S
/
r r
O O O
/ I ~o
O NJ
i
O
(132)
(2- (2-Methoxy-phenylsulfanyl) -phenyl J -acetic acid
2-Methoxythiophenol (2.8g, 20mmo1) was added to a solution of
potassium hydroxide (4.6g, 80mmo1) in water (50m1) and the
mixture was degassed for 15 minutes. 2-Iodophenylacetic acid
(5.248, 20mmo1) and copper bronze (64mg, lmmol) were then
added to the reaction mixture, which was refluxed overnight.
The solution was cooled down, filtered and the precipitate
washed with water (50m1). The filtrate was acidified with conc
HCl (pH 1), extracted with dichloromethane (3x100m1). The
organics were combined, extracted with saturated brine, dried
over sodium sulphate and evaporated in vacuo to give the title
compound as a pale brown oil which solidified overnight. The
compound was used without any further purification (5.10g,
930) .
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6-Methoxy-I1H-dibenzo[b,fJthiepin-10-one
[2-(2-Methoxy-phenylsulfanyl)-phenyl]-acetic acid (5.408,
20mmol) was dissolved in methanesulfonic acid (50m1) and the
mixture was heated for 2 hours at 90°C under stirring and a
nitrogen atmosphere. The reaction mixture was cooled to room
temperature and poured onto ice with stirring. The black
precipitate was filtered and dried over night in a vacuum oven
(50°C). The compound was used without any further purification
( 4 . 60g, 91 0 ) .
6-Hydroxy-lIH-dibenzo(b,fJthiepin-10-one
6-Methoxy-11H-dibenzo[b,f]thiepin-10-one (1.548, 6mmo1) and
pyridine hydrochloride (10g) were heated for 2 hours at 200°C
with stirring and NZ atmosphere. The reaction was cooled down
to room temperature and then triturated in water (200m1) The
pale green precipitate was filtered and dried overnight in a
vacuum oven (50°C) (1.408, 960) . 1HNMR (300MHz, DMSO-d6) : 8H=
4.20 (2H, s), 7.05-7.69 (7H, m), 10.56 (1H, s).
Trifluoro-methanesulfonic acid 11-oxo-10,11-dihydro
dibenzo(b,fJthiepin-4-yl ester
6-Hydroxy-11H-dibenzo[b,f]thiepin-10-one (242mg, lmmol) was
dissolved in dry pyridine (5ml) and trifluoromethanesuphonic
anhydride (0.17m1, lmmol) was added drop wise to the stirred
solution at 0°C under NZ atmosphere. The reaction mixture was
left to react for 4 hrs and was then poured into water. (50m1)
The organic were extracted with dichloromethane (3x50m1),
washed with 0.2N HC1, dried over magnesium sulphate and
evaporated in vaccuo to give a dark brown solid. This solid
was purified by column chromatography (dichloromethane/hexane,
3:7, Rf=0.15) to give the title compound as a pale brown solid
(0.37g, 1000) . 1HNMR (300MHz, DMSO-d6) : bH= 3.70 (2H, s) , 7.31-
7.8 (7H, m) .
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6-(4,9,5,5-Tetramethyl-(1,3,2]dioxaborolan-2-yl)-11H-
dibenzo(b,f]thiepin-10-one
Trifluoro-methanesulfonic acid 11-oxo-10,11-dihydro-
dibenzo[b,f]thiepin-9-yl ester (0.374 g, lmmol),
bis(pinacolato)diboron (305mg, l.2mmo1) and potassium acetate
(294mg, 3mmo1) were dissolved in 1,4-dioxane (5mL) and the
mixture was degassed for 5 min. Pd(dppf)C12 (40mg, 0.05mmo1)
and dppf (28mg, 0.05mmo1) were added to the vessel, and the
reagents heated to 100°C under nitrogen with stirring for 12
hours. The reaction mixture was purified by flash
chromatography (dichloromethane/hexane, 1:4) and the black
residue was used without further purification (0.35g).
2-Morpholin-4-yl-6-(11-oxo-l0,ll-dihydro-dibenzo(b,f]thiepin-
4-yl) -pyran-4-one (132)
2-Chloro-6-morpholin-4-yl-pyran-4-one (3)(215mg, lmmol), 6-
(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-11H-
dibenzo[b,f)thiepin-10-one (352mg, lmmol), and ground
potassium carbonate (276mg, 2mmo1) were suspended in 1,9-
dioxane (10m1) and degassed for 5 minutes. Pd(PPh3)4 (57mg,
0..05mmo1) was then added and the reaction mixture was then
heated at 90°C for 4hours under a vigorous stirring and a NZ
atmosphere. The solvent was removed in vaccuo and the residue
was then suspended in water (100m1). The organics were
extracted with dichloromethane (3x100m1), combined, washed
with saturated brine and dried over sodium sulphate. The
solvent was removed in vaccuo and the residue was purified by
column chromatography (silica; ethyl acetate:ethanol; 9:1) to
give the title compound as a pale brown solid (0.128, 290).
1HNMR (300MHz, DMSO-d6) : 8H= 3.40 (9H, t) , 3.70 (4H, t) , 4. 43
(2H, s), 5.55 (1H, d), 6.29 (1H, d), 7.25-7.55 (5H, m), 7.78-
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7.81 (1H, m), 8.20-8.22 (1H, m); m/z (LC-MS, ESP), RT=4.12
min, (M++1)= 406.
Example 13: 2-(10,11-Dihydro-dibenzo[b,f]thiepin-4-yl)-6-
morpholin-4-yl-pyran-4-one
~O _
O OH _
S ~ ~ ~ S ~ ~ ~ S
/ ~ / --
O
O
OTf _ O~B~O _
S \ / \ S
(133)
4-Methoxy-10,11-dihydro-dibenzo(b,fJthiepine
Hydrazine hydrate (4ml) and potassium hydroxide (2.728,
48mmo1) was added to 6-methoxy-11H-dibenzo[b,f]thiepin-10-one
(4.lOg,-l6mmol) in ethylene glycol (20m1) and the reaction
mixture was heated at 175°C for 3 hours. The reaction mixture
was cooled down to room temperature and water was added
(100m1). The white solution was extracted with ether
(3x200m1), the organics were combined, washed with water
(100m1), brine (100m1) and dried over magnesium sulphate. The
solvent was removed in vacuo to give an oil which solidify
upon standing to give a brown solid which was used without any
further purification (2.458, 630).
10,11-Dihydro-dibenzo(b,fJthiepin-~-of
4-Methoxy-10,11-dihydro-dibenzo[b,f]thiepine (2.428, lOmmol)
and pyridine hydrochloride (15g) were heated with stirring at
180°C for one hour. Water (100m1) was added to the reaction
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mixture and the organics were extracted with ethyl acetate
(3x100 ml). The organics were combined and washed with 2N HC1
(50m1), brine (50 ml), and dried over magnesium sulphate. The
solvent were removed in vacuo and the residue was purified by
column chromatography (1:9; dichloromethane:hexane) to give
the desired compound as a white solid (1.558, 680). 1HNMR
(300MHz, DMSO-d6) : 8H= 3.13-3.23 (4H, m) , 6.70 (2H, t) , 6. 97-
7.15 (4H, m), 7.38 (lH,s) 9.78 (1H, s); m/z (LC-MS, ESP),
RT=4.59 min, (M++1)= 229.
Trifluoro-methanesulfonic acid 10,11-dihydro-
dibenzo(b,f]thiepin-4-yl ester
10,11-Dihydro-dibenzo[b,f]thiepin-4-of (1.26g, 5.5mmo1) was
dissolved in dry pyridine (5m1) and trifluoromethanesuphonic
anhydride (1.12m1, 6.6mmo1) was added drop wise to the stirred
solution at 0°C under Nz atmosphere. The reaction mixture was
left to react for 4 hrs and was then poured into water.
(100m1) The organic were extracted with dichloromethane
(3x50m1), washed with 0.2N HC1, dried over magnesium sulphate
and evaporated in vaccuo to give a dark brown solid. This
solid was purified by flash chromatography (dichloromethane)
to give an oil (1.1g, 56%). 1HNMR (300MHz, DMSO-d6): 8H= 3.25-
3.29 (2H, m), 3.37-3.41 (2H, m), 7.12-7.17 (1H, m), 7.21-7.31
(3H, m), 7.38-7.41 (3H,s).
2- (10, 11-Dihydro-dibenzo (b, f) thiepin-4-yl) -~, ~, 5, S
tetramethyl-(1,3,2Jdioxaborolane
Trifluoro-methanesulfonic acid 10,11-dihydro-
dibenzo[b,f]thiepin-4-yl ester (1.088, 3mmo1),
bis(pinacolato)diboron (914mg, 3.6mmo1) and potassium acetate
(883mg, 9mmo1) were dissolved in 1,4-dioxane (lOmL) and the
mixture was degassed for 5 minutes. Pd(dppf)C12 (121mg,
0.15mmo1) and dppf (83mg, 0.15mmo1) were added to the vessel,
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and the reagents heated to 100°C under nitrogen with stirring
for 12 hours. The reaction mixture was purified by flash
chromatography (dichloromethane) and the black residue was
used without further purification (0.878).
2-(l0,ll-Dihydro-dibenzo(b,fJthiepin-4-yl)-6-morpholin-4-yl-
pyran-4-one
2-Chloro-6-morpholin-9-yl-pyran-4-one (3)(1.128, 5.2mmo1), 2-
(10,11-Dihydro-dibenzo[b,f]thiepin-4-yl)-4,4,5,5-tetramethyl-
[1,3,2]dioxaborolane (880mg, 2.6mmo1), and ground potassium
carbonate (720mg, 5.2mmo1) were suspended in 1,4-dioxane
(10m1) and degassed for 5 minutes. bis(tri-t-
butylphosphine)palladium (66mg, 0.13 mmol) was then added and
the reaction mixture was then heated at 90°C for 4 hours under
a vigorous stirring and a NZ atmosphere. The solvent was
removed in vaccuo and the residue was then suspended in water
(100m1). The organics were extracted with dichloromethane
(3x100m1), combined, washed with saturated brine and dried
over sodium sulphate. The solvent was removed in vaccuo and
the residue was purified by column chromatography (silica;
ethyl acetate: ethanol; 9:1) to give a pale brown solid (50mg,
50) . 1HNMR (300MHz, DMSO-d6) : 8H= 3.24-3.32 (6H, m) , 3.44 (2H,
t), 3.66 (4H, t), 5.50 (1H, d), 6.10 (1H, d), 7.08-7.51 (7H,
m); m/z (LC-MS, ESP), RT= 4.48min, (M++1)= 392.
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Example 14: 2-Morpholin-4-yl-6-(lOH-phenothiazin-4-yl)-pyran-
4-one
~O O
I \ N I \ I \ N I \ ~ I \ N I
/ / / S / / S /
OH OH OH
O O ~O O
/ ~O
\ N \ O
I I ~ I / I / ~ O~N \ O NJ
/ S / S S I
O;S o F O~B\O I / O
O~ ~F
F (134)
/I ~o
HN \ O NJ
S I I
/ O
(135)
10H-Phenothiazin-4-o1
To a solution of 3-phenylamino-phenol (5 g, 26.99mmo1) in 1,2-
dichlorobenzene (50 ml) was added S$ sulfur(1.82 g, 56.76 mmol)
in a single portion and iodine (0.1 g, 0.39 mmol) which was
added in three portions over 10 minutes. A reflux condenser
was attached to the reaction vessel which was heated to 185°C
under a nitrogen atmosphere. The mixture was stirred at this
temperature for 4 hours and then allowed to cool to room
temperature. The reaction mixture was filtered to remove a
black precipitate and the filtrate diluted with Et20 (100 ml)
and washed with water (2x100 ml). The organic layer was
separated and the volatile solvents removed to give a deep
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green oil that was purified by flash column chromatography
(Si02) (Hexanes then 8:1-Hexanes:EtOAc) to give a pale yellow
solid (2.38 g, 40.96%) m/z (LC-MS, ESP) 216 [M+H]+, R/T = 9.12
mlns.
4-Hydroxy-phenothiazine-10-carboxylic acid tert-butyl ester
To a solution of 10H-Phenothiazin-4-of (0.77 g, 3.58 mmol) in
anhydrous pyridine (10 ml) was added di-tertiary butyl
dicarbonate (3.12 g, 14.31 mmol) in a single portion. The
solution was heated to 80°C and stirred under a nitrogen
atmosphere for 60 minutes before being allowed to cool to room
temperature and treated with water (20 ml) and extracted with
EtOAc (2x30 ml). The organic layers were then washed with
water (20 ml), dried using MgS09, filrtered and concentrated in
vacuo to give an amber oil. The crude residue was treatyed
with MeOH (15 ml) and solid NaOH (0.65 g, 16.25 mmol). The
mixture was heated to 80°C for 60 minutes then cooled to room
temperature and neutralised to pH7 with 1M HC1 solution. The
resulting suspension was then filtered and dried to give the
title compound as a beige solid (1.13 g, 1000) that was used
without further purification. m/z (LC-MS, ESP): 315 [M-H]-, R/T
- 4.72 mins.
4-Trifluoromethanesulfonyloxy-phenothiazine-10-carboxylic acid
tert-butyl ester
Trifluoromethanesulfonic anhydride (2.95 ml, 17.09 mmol) was
added in a dropwise fashion over 10 minutes to a cooled (0°C)
stirred solution of 4-Hydroxy-phenothiazine-10-carboxylic acid
tert-butyl ester (3.60 g, 11.41 mmol) in pyridine (40 ml).
The reaction mixture was stirred at 0°C for 1 hour before the
addition of water (80 ml). The mixture was extracted using
EtOAc (2x60 ml). The organic extracts were then dried using
MgSOq, filtered and concentrated in vacuo to give a dark brown
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oil. The crude residue was then purified by flash
chromatography (Si02) (4:1-Hexanes:EtOAc) to yield a yellow oil
(5.02 g, 98.290) m/z (LC-MS, ESP): 348 [M+H-BOC]+, R/T = 5.61
mins
4-(4,9,5,5-Tetramethyl-(1,3,2Jdioxaborolan-2-yl)-
phenothiazine-10-carboxylic acid tert-butyl ester
To a stirred solution of 4-trifluoromethanesulfonyloxy-
phenothiazine-10-carboxylic acid tert-butyl ester (3.0 g, 6.7
mmol) in anhydrous dioxane (10 ml) was added
bis(pinacolato)diboron (2.05 g, 8.06 mmol) and potassium
acetate (1.96 g, 20.01 mmol). The reaction was then degassed
(sonication for 20 minutes then saturated with N2) before the
addition of dichloro[1,1'-bis(diphenylphosphino)ferrocene]
palladium(II) dichloromethane adduct (0.27 g, 0.33 mmol). The
reaction mixture was degassed for a further 20 minutess before
a reflux condenser was attached to the reaction vessel which
was then heated to 90°C and stirred vigorously for 72 hours.
The dark brown reaction mixture was then allowed to cool to
room temperature before it was applied to a thick silica pad
prepared in hexanes and eluted with hexanes:CH2C12-(2:1). The
eluent was concentrated in vacuo to give a dark brown oil
(2.85 g, 1000) that was used for the next transformation with
no further purification. m/z (LC-MS, ESP): 326 [M+H-BOC]+, R/T
- 5.86 mins
4-(6-Morpholin-4-y1-4-oxo-4H-pyran-2-yl)-phenothiazine-10
carboxylic acid tert-butyl ester (134)
Powdered potassium carbonate (2.03 g, 14.68 mmol) and 2-
Chloro-6-morpholin-4-yl-pyran-4-one (1.44 g, 6.70 mmol) were
added to a stirred solution of 4-(4,4,5,5-tetramethyl-
[1,3,2]dioxaborolan-2-yl)-phenothiazine-10-carboxylic acid
tert-butyl ester (2.85 g, 6.70 mmol) in anhydrous dioxane (20
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ml) and the mixture degassed (sonication for 20 minutes then
saturated with NZ) thoroughly. Tetrakis (triphenylphosphine)
palladium was then added in a single portion and the mixture
degassed (sonication for 20 minutes then saturated with Nz)
once again before a reflux condenser was attached and the
mixture heated to 100°C under a nitrogen atmosphere for 20
hours. Water (30 ml) was added and the mixture extracted with
EtOAc (3x30 ml). The organic extracts were then dried using
MgS09, filtered and concentrated in vacuo to yield a dark
brown, crystalline solid (3.21 g, 1000) that was taken forward
with no further purification. m/z (LC-MS, ESP): 479 [M+H]+, R/T
- 4.55 mins
2-Morpholin-9-y1-6- (l OH-phenothiazin-4-yl) -pyran-9-one (135)
To a stirred solution of 9-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-
yl)-phenothiazine-10-carboxylic acid tert-butyl ester (3.65 g,
7.63 mmol), in CHZC12 (30 ml) was added trifluoroacetic acid in
a single portion. The mixture was stirred at room temperature
for 20 hours whereupon the reaction was concentrated in vacuo
to give a thick syrup that was basified in a dropwise fashion
with saturated NaHC03 (40 ml). The dark green mixture was then
stirred at room temperature for 18 hours. The mixture was
filtered and the filtrant retained, washed with water and
dried to give the title compound as a dark green solid (2.898,
83.740 over 3 steps) m/z (LC-MS, ESP): 479 [M+H]+, R/T = 4.05
mans
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Example 15: 4-Morpholin-4-yl-6-thianthren-1-yl-pyran-2-one
O O O
w s I w I w S I w I w S I w
a ~ - . . -- a / ----
a
S s s
~O
O S O S O NJ
WSWSH WSWs1 \S ,s
/ / /
/
s S a /
O \ N J / ~ ~O
\ / N J
\ S \ S S
/ ~ ~ \ S O
S I / O
Thianthrene-1-carboxylic acid
t-Butyl lithium (1.7M in hexane, 55.1m1, 93.6mmo1) was added
drop wise to a stirred solution of thianthrene (16.98, 78mmo1)
in dry THF (250m1) at -78°C over 30 minutes under an inert
atmosphere (N2). The reaction mixture was allowed to warm to
room temperature and the resulting reddish solution was left
stirring for 24 hours. The mixture was then cooled down to -
78°C and carbon dioxide (from dry ice pellet and dried by
passing over some activated A4 sieves) was bubbled into the
solution for 1 hour. The reaction was warmed up back to room
temperature with COZ still bubbling through it for another
hour. Water (10m1) was then added carefully to the solution
and the pH was adjusted to 1 (pH paper) with 2N HC1. The
solvent was removed in vacuo and the yellow solid formed was
filtered and dried overnight in a vacuum desiccators. The
solid was then recrystalised from methanol to give the desired
product as a pale yellow crystalline solid (11.98, 590). 1HNMR
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(300MHz, CDC13): 8H = 7.25 (3H, m); 7.50 (4H, m). m/z (LC-MS,
ESP) : RT= 4.53min, (M- - 1)= 259
I-Thianthren-1-yl-ethanone
Methyl lithium (1.6M in ether, 57m1, 90mmo1) was added drop
wise to a stirred solution of thianthrene-1-carboxylic acid
(11.718, 45mmol) in dry tetrahydrofuran (200m1) at -78°C over
30 minutes under an inert atmosphere (NZ). The reaction mixture
was allowed to warm to room temperature and (very thick white
suspension present) was left stirring for 4 hours. Water
(10m1) was then added carefully to the solution and the pH was
adjusted to 1 (pH paper) with 2N~HC1. The solvent was removed
in vacuo and the yellow solid formed was filtered and dried
overnight in a vacuum desiccators. The solid was then purified
by column chromatography (ethyl acetate/hexane; 1:9) and was
recrystalised from ethanol to give the desired product (6.588,
570). 1HNMR (300MHz, CDC13): 8H = 2.65 (3H, s); 7.26 (3H, m);
7.47 (2H, m); 7.62 (2H, d). m/z (LC-MS, ESP) RT= 9.95 min; (M+
+ 1)= 259
3-Oxo-3-thianthren-I-yl-dithiopropionic acid
A solution of CSz (1.55m1, 25.5mmo1) and 1-thianthren-1-yl-
ethanone (6.598, 25.5mmo1) in dry tetrahydrofuran (20m1) was
added drop wise to a solution of potassium t-butoxide (5.738,
51mmo1) in dry tetrahydrofuran (50m1) under NZ at 0°C. A red
coloration and the formation of a precipitate were observed.
The mixture was left under vigorous stirring over the weekend
and was then poured onto water (200m1) and extracted with
ether (3x100m1). The aqueous was acidified with 2N HZSO9 to pH
1 (Whatmann pH paper) and the extracted with ether (3x100m1).
The organic were dried over magnesium sulphate and the solvent
was evaporated in vacuo to give the desired product as a dark
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orange resin (S.OOg, 590). m/z (LC-MS, ESP), RT= 5.11; (M -1)=
333
3-Oxo-3-thianthren-1-y1-dithiopropionic acid ethyl ester
Tetrabutylammonium hydrogen sulphate (5.1g, l5mmol) and sodium
hydroxide (1.2g, 30mmo1) were dissolved in water (50m1) A
solution of 3-oxo-3-thianthren-1-yl-dithiopropionic acid
(5.02g, 15mmo1) in dichloromethane (50m1) was added to the
solution in one portion and was stirred vigorously for 30
minutes. The aqueous layer was removed and iodoethane (9m1)
was added to the dichloromethane solution that was then
stirred for lhr. The solvent was removed in vacuo and the
residue was taken into water (200m1) The organics were
extracted with ether (3x100m1), dried over magnesium sulphate
and evaporated in vacuo. The residue was then purified by
column chromatography (ethyl acetate:hexane; 1:4) to give the
desired compound as a bright yellow solid (4.00 g, 730). 1HNMR
(300MHz, CDC13) : 8H = 1. 43 (3H, t) , 3.33 (3H, q) , 6.57 (1H, s) ,
7.26 (3H, m), 7.51 (3H, m), 7.60 (1H, m), 15.09 (1H, s); m/z
(LC-MS, ESP), RT=6.50 min, (M--1)= 361.
3-Morpholin-4-y1-I-thianthren-1-y1-3-thioxo-propan-1-one
Morpholine (0.96m1, 11mmo1) was added to a solution of 3-oxo-
3-thianthren-1-yl-dithiopropionic acid ethyl ester (3.998,
llmmol) in ethanol (20m1). The reaction was refluxed for 8
hours and was then cooled to room temperature. The precipitate
formed was filtered and dried to give the desired product as a
bright orange solid (3.508, 820). m/z (LC-MS, ESP), RT=
4.81and 5.33min same (M+1)= 388
3-Ethylsulfanyl-3-morpholin-9-y1-1-thianthren-1-y1-propenone
3-Morpholin-4-yl-1-thianthren-1-yl-3-thioxo-propan-1-one
(3.498, 9mmo1), iodoethane (0.8m1, lOmmol), and grinded
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potassium carbonate (1.388, l0mmol) were suspended in acetone
(20m1) and the mixture was refluxed for 24 hours. The solvent
was removed in vacuo and the residue was taken into water
(50m1). The organics were extracted into dichloromethane
(3x100m1), dried over magnesium sulphate and evaporated in
vacuo. The crude product was purified by column chromatography
(ethyl acetate/hexane) to give the desired product as a yellow
solid (2.268, 600) . 1HNMR (300MHz, CDC13) : 8H= 1.34 (3H, t) ,
2.96 (2H, q), 3.73 (4H, m), 3.84 (4H, m), 7.21 (3H, m), 7.47
(4H, m) ; m/z (LC-MS, ESP) , RT= 5.01min, (M++1)= 416.
4-Morpholin-4-y1-6-thianthren-1-yl-pyran-2-one (136)
A suspension of activated zinc dust (0.658, l0mmol), ethyl
bromoacetate (0.56m1, 5mmo1) and a few crystals of iodine in
dry tetrahydrofuran (20m1) were heated at 50°C for one hour
with stirring under a Nz atmosphere. A solution of 3-
ethylsulfanyl-3-morpholin-4-yl-1-thianthren-1-yl-propenone
(1.048, 2.5mmo1) in dry tetrahydrofuran (20m1) was added drop
wise with stirring and the mixture was refluxed for 12 hours
under a NZ atmosphere. The mixture was then poured over ice
cold dilute 3% HZSO9 (50m1), the aqueous layer was extracted
with ethyl acetate (3x50m1), the combined extracts were dried
over magnesium sulphate and the solvent was evaporated in
vacuo. The residue was purified by column chromatography
(ethyl acetate/hexane) to give the desired product (0.35g,
350) . 1HNMR (300MHz, CDC13) : bH= 3.45 (4H, t) , 3.85 (4H, t) ,
5.35 (1H, d) , 6.29 (1H, d) , 7.26 (3H, m) , 7.50 (3H, m) 7. 61
(1H, m); m/z (LC-MS, ESP), RT= 9.50min, (M++1)= 396.
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Example 16: 6-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-
thianthrene-2-carboxylic acid amide Derivatives
COOH COOH COOH
\ \ \ /
Br
soZci I / so2H
HOOC~SOzH
Br O ~O
Br
S
\ \ S S
HO I / S I / p I / I / O I / I /
g i ~ ~S
O O O
/ I ~o / I ~o
\ O NJ S \ O NJ
O I / O Na0 ( / O
O O
(137) (138)
3-Chlorosulfonyl-4-fluoro-benzoic acid
Chlorosulphonic acid (100 ml, 1.5 mol) was gradually added to
4-fluorobenzoic acid (438, 0.307mo1) with stirring. The clear
dark yellow mixture was heated to 150°C for 24 hours. The
yellow solution was cooled back to room temperature and poured
onto ice with vigorous stirring. The white precipitate was
filtered and pressed dry. The solid was dried overnight in a
desiccator under vacuum and over activated silica (54.658,
750). Mp: 116-117°C; m/z (LC-MS, ESP), RT= 4.03min, (M--1)=
237-239 (ratio 1:3).
9-Fluoro-3-sulfino-benzoic acid
Sodium sulphite (1308, 1.034mo1) was added slowly to a
solution of 3-chlorosulfonyl-4-fluoro-benzoic acid (49.398,
0.207mo1) in water (150m1) at 0°C with a vigorous stirring.
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After the addition was completed the reaction was warmed back
to room temperature for 1 hour and the pH of the solution was
kept around pH 6-7 with 2N sodium hydroxide solution. The
white milky suspension was filtered and the solid washed with
2N sodium hydroxide solution (150m1) and then water (100m1).
The filtrate was then cooled in an ice bath and concentrated
HC1 was added until no more precipitate was formed (pH<1). The
white precipitate was then filtered, pressed dry and left in a
dessicator overnight under vacuum and over activated silica
(27. 92g, 660) . m/z (LC-MS, ESP) , RT= 0.98min, (M--1)= 203
4- (2-Bromo-phenylsulfanyl) -3-sulfino-benzoic acid
2-Bromobenzenethiol (25g, 132 mmol) was added to a solution of
4-fluoro-3-sulfino-benzoic acid (13.58, 66mmo1) and NaOH
pellets (11g, 264mmo1) in water (30m1). The yellow mixture was
then degassed for 10 minutes and then heated to 140°C for 48
hours. The reaction was then cooled to 0°C and acidified to pH
4-5 (pH paper) with concentrated HC1. The precipitate formed
was filtered, washed with hexane and was dried in a vacuum
dessicator over activated silica overnight (20.698, 840). m/z
(LC-MS, ESP), RT= 3.67min, (M -1)= 373.
6-Bromo-thianthrene-2-carboxylic acid
4-(2-bromo-phenylsulfanyl)-3-sulfino-benzoic acid (148,
38mmo1) was added slowly to a stirred solution of
methanesulphonic acid (160m1). The purple solution was heated
to 60°C for 3 hours. The reaction was cooled down to room
temperature and was poured into ice (300m1) where an off-white
precipitate appeared. The solid was filtered and washed with
water (100m1) and then dried in a vacuum dessicator over
activated silica (9.488, 730). 1HNMR(300MHz, CDC13): 7.29
8H=
(1H, t) 7.59 (1H, dd) , 7.70 (lH,dd)7.74 (1H, d) , 7.87 (1H,
,
dd), 8.03 (1H, d).m/z (LC-MS, ESP), RT= 4.99min, (M--1)=339
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6-Bromo-thianthrene-2-carboxylic acid methyl ester
To 6-bromo-thianthrene-2-carboxylic acid (9g, 28mmo1) in
methanol (180m1) was slowly added conc. HzS09 (5 ml). The milky
white suspension was heated to 80°C until all the solid had
gone into solution (2hrs). The suspension was concentrated in
vacuo. Water (100m1) was added and the organics were then
extracted with dichloromethane (3 x 70 ml), dried over MgS04
and evaporated in vacuo, yielding to a yellow solid. (4.488,
95 0) . 1HNMR (300MHz, CDC13) : ~H= 3. 94 (3H, s) ; 7. 13 (1H, t) ,
7.94 (1H, dd), 7.54 (lH,dd) 7.61 (1H, d), 7.93 (1H, dd), 8.13
(1H, d).
6-(4,9,5,5-Tetramethyl-(1,3,2]dioxaborolan-2-yl)-thianthrene-
2-carboxylic acid methyl ester
6-Bromo-thianthrene-2-carboxylic acid methyl ester (1g,
2. 8mmo1) , bis (pinacolato) diboron (0.868, 3. 4mmol) and
potassium acetate (0.12g, 0.14mmo1) in 1,4-dioxane (15m1) was
degassed for 15 minutes. To the yellow suspension was then
added PdCl2(dppf) (78mg, 0.14mmo1) and dppf (0.83g, 8.5mmo1).
The dark red mixture was heated to 90°C under a N2 atmosphere
for 48 hours. The crude mixture was purified by flash
chromatography (dichloromethane) to give viscous brown oil
(1.13g), which was used without any further purification.
6-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-thianthrene-2-
carboxylic acid methyl ester (137)
6-(4,9,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-thianthrene-
2-carboxylic acid methyl ester (1.1g, 2.83mmo1), 2-chloro-6-
morpholin-4-yl-pyran-4-one (0.738, 3.4mmo1) and KzC03 (0.8g,
5.66mmo1) were dissolved in dry 1,4-dioxane (7m1). The mixture
was degassed for 15 mins and Pd(PPh3)9 (0.16 g, 5 mol o) was
then added The dark brown mixture was heated to 90°C under an
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atmosphere of Nz for 24 hour. The reaction mixture was
concentrated in vacuo and water (100m1) was added. The brown
solid was filtered and washed with water (1.238, 960). m/z
(LC-MS, ESP), RT= 4.49 min, (M++1)= 454.
6-(6-Morpholin-4-y1-4-oxo-~H-pyran-2-yl)-thianthrene-2-
carboxylate sodium salt (138)
6-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-thianthrene-2-
carboxylic acid methyl ester (l.lg, 2.43mmo1) and NaOH Pellets
(97mg, 2.43mmo1) were dissolved in methanol (40m1). The brown
suspension was heated to 80°C under NZ for 24 hours. The
solvent was removed in vacuo and the residue was triturated
with diethyl ether. The product was collected by filtration as
a fine dark brown powder (1.118, 990). m/z (LC-MS, ESP),
RT=3.90 min, (M- -1)= 438
6-(6-Morpholin-4-y1-4-oxo-4H-pyran-2-yl)-thianthrene-2-
carboxylic acid amide Derivatives
6-(6-morpholin-4-yl-4-oxo-4H-pyran-2-yl)-thianthrene-2-
carboxylate sodium salt (138)(20mg, 0.04mmo1), HBTU (l8mg,
0.05mmo1), di-isopropylethylamine (9u1, 0.05mmo1), the
appropriate amine (0.04mmo1) and dry dimethylacetamide
(0.5m1). The dark brown mixture was stirred at room temp for 2
hours and then purified by preparative HPLC to give the
desired products, which are shown below:
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~o
o NJ
o ~ ~ o
R
Compound R Purity Retention Time M++1
(Mins)
139 ~N\ 95 3.65 453
140 I 95 3.7 467
,Ny
141 ~ 95 4.11 495
~N~
142 ~N\, 95 3.78 467
143 ~,"~\= 95 4.28 495
14 4 ~N\, 95 4 . 03 9 81
195 H 90 4.22 495
N~
14 6 N\f 95 4 . O1 4 81
147 NC~N~t 85 3.84 477
14 8 F3~~N\. 90 4 . 1 521
149 90 3.81 523
H
O N~.
150 ~ 95 4.1 519
~ H
O Nw.
151 ,"~\ 95 4 .54 521
152 N 95 4.1 493
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153 N\= 95 4.12 493
154 a 95 4.77 535
H
N~
155 N\y 95 4.3 507
156 95 3.89 493
N~
157 95 4.14 507
N~
158 95 4.00 537
0
~N~
159 95 4.02 537
0
~N~
160 0~ 95 3.69 509
~N~.
161 95 4.31 521
N~
162 ° H 95 3.73 558
\ 'N~.
I N
H
163 ,N\ 95 3.72 552
N
O
164 ~ ,N\. 95 3.12 537
/N J
165 ~N,~",\, 95 3.49 524
of
166 N\. 95 3.29 550
GN
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167 N\ 95 3.23 536
N~ '
G
168 N\ 95 3.22 550
N
169 N\ 95 3.62 572
I
,N~
170 N\ 95 3.43 530
~ ~N
I
171 N\ 95 3.27 530
N
172 ,"~\ 95 3.21 530
y
\N
173 N\ N\ 95 3.26 544
174 I 95 3.3 558
N~N~
I~
175 ~N~N\. 95 3 . 05 57 9
~NJ
176 ~r",\ 95 3.17 533
N \ '
~NH
177 ~N~N~' 95 3 . 19 552
of
178 0~ H 95 3.18 566
~N~N~
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179 ~ ~,"J\. 95 3.44 641
I
180 ~N~ 95 3.49 651
'
~N
r
O~'N
J
~O
181 N 95 3.29 538
~N~
J
182 ~ 95 3.27 552
H
~N~N~,
B) Biological Examples
Materials and Methods
In vitro ATM inhibition Assays
In order to assess the inhibitory action of the compounds
against ATM in vitro, the following assay was used to
determine ICso values .
ATM protein was immunoprecipitated from HeZa cell nuclear
extract using rabbit polyclonal anti-sera raised to the C-
terminal 500 amino-acid residues of the human ATM protein.
The immunoprecipitation was performed according to the
methodology described by Banin, S. et al. (1998). 10 ~1 of
immunoprecipitated ATM in Buffer C (50 mM Hepes, pH 7.9, 6 mM
MgCl2, 150 mM NaCl, 0.1 mM sodium orthovanadate, 4 mM MnCl2,
0.1 mM dithiothreitol, 10% glycerol) was added to 32.5 ul of
buffer C containing 1 ug of the ATM substrate GSTp53N66 in a
V-bottomed 96 well polypropylene plate. The GSTp53N66
substrate is the amino terminal 66 amino acid residues of
human wild type p53 fused to glutathione S-transferase. ATM
phosphorylates p53 on the residue serine 15 (Banin, S. et al.
(1998)). Varying concentrations of inhibitor were then added.
All compounds were diluted in DMSO to give a final assay
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concentration of between 100pM and 1 nM, with DMSO being at a
final concentration of lo. After 10 minutes of incubation at
37°C, the reactions were initiated by the addition of 5 ul of
500 ~M Na-ATP. After 1 hour with shaking at 37°C, 150 u1 of
phosphate buffered saline (PBS) was added to the reaction and
the plate centrifuged at 1500 rpm for 10 minutes. 5 pl of the
reaction-was then transferred to a 96 well opaque white plate
containing 45 ul of PBS to allow the GSTp53N66 substrate to
bind to the plate wells. The plate was covered and incubated
at room temperature for 1 hour with shaking before discarding
the contents. The plate wells were washed twice by the
addition of PBS prior to the addition of 30 (w/v) bovine serum
albumin (BSA) in PBS. The plate was incubated at room
temperature for 1 hour with shaking before discarding the
contents and washing twice with PBS. To the wells, 50 ul of a
1:10,000 dilution of primary phosphoserine-15 antibody (Cell
Signaling Technology, #9284L) in 3o BSA/PBS was added to
detect the phosphorylation event on the serine 15 residue of
p53 elicited by the ATM kinase. After 1 hour of incubation at
room temperature with shaking, the wells were washed four
times with PBS prior to the addition of an anti-rabbit HRP
conjugated secondary antibody (Pierce, 31462) with shaking for
1 hour at room temperature. The wells were then washed four
times with PBS before the addition of chemiluminescence
reagent (NEN Renaissance, NEL105). The plate was then shaken
briefly, covered with a transparent plate seal and transferred
to a TopCount NXT for chemiluminescent counting. Counts per
second, following a one second counting time, were recorded
for each reaction.
The enzyme activity for each compound is then calculated using
the following equation:
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o Inhibition =100- (cpm of unknown - mean negative cpm)x100
(mean positive cpm - mean negative cpm)
Sensitisation of Cells to ionising radiation or DNA double
strand break chemotherapies
To test the efficacy of the ATM inhibitor compound 4 on its
ability to sensitise cells to ionising radiation or~to DNA
double strand break inducing chemotherapeutics, clonogenic
survival assays were performed using the HeLa or LoVo human
tumour derived cell lines. The HeLa line was used for
ionising radiation studies while LoVo was used for studies
with chemotherapeutic agents. Enough cells to give 100
colonies per treatment were seeded into 6 well dishes 4-6
hours prior to the addition of compound 4 at the
concentrations shown on the graphs. After 1 hour, a
concentration range of either etoposide (figure 2),
camptothecin (figure 3) or doxorubicin (figure 4) was added.
For ionising radiation treatment (figure 1), after 1 hour of
incubation with compound 4, cells were irradiated at 1Gy/min
using a Faxitron 43855D X-ray cabinet. For all treatments,
after a further 16 hours incubation, drug containing media was
removed and fresh media added prior to a further incubation of
10 days before the staining of colonies with Giemsa. All
compounds were solubilised in DMSO, with a final concentration
on cells of no more than O.lo. Resulting colonies containing
>50 cells were counted as positives.
Recombinant retroviral vectors and virus preparation.
The ~'-/LTR-/Vpr- replication deficient HIV-1 gag/pol expressing
packaging constructs were designed based on the vector LOP2GPH
(Haselhorst et al., 1998 Development of cell lines stably
expressing human immunodeficiency virus type 1 proteins for
studies in encapsidation and gene transfer. J Gen Virol, 79,
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231-7.). A HIV-1 integrase mutant packaging construct, which
codes for a D64V amino acid change in the integrase gene, was
made by site directed mutagenesis (Quikchange mutagenesis
system, Stratagene). The HIV-1 luciferase transfer vector,
HIV-Luc, was constructed by inserting the firefly luciferase
gene in-between two HIV-1 LTR sequences and a ~I' HIV-1 RNA
packaging signal sequence. The VSV G envelope expression
plasmid has been described previously (Naldini et al., (1996)
In vivo gene delivery and stable transduction of nondividing
cells by a lentiviral vector. Science, 272, 263-7). HIV-1
recombinant retroviral stocks were produced using a
modification of the three-plasmid expression system described
by Naldini et al., 1996. 6 x106human kidney 293T cells were
co-transfected with 10 ug packaging construct WT or integrase
D64V mutant, 8 ug HIV-Luc transfer vector and 5 ~g VSV G
envelope protein expression plasmids using Lipofectamine-2000
reagent (Gibco-BRL). 48 hours post transfection retrovirus-
containing cell culture supernatants were harvested, filtered
through 0.45 uM cellulose acetate membranes and stored at -
80°C. Recombinant HIV-1 viral titres were estimated using the
HIV-1 p24 gag antigen ELISA kit from Beckman-Coulter,
according to the manufacturers' instructions.
Retroviral transductions (LUCIA).
For the HIV-1 based luciferase assays (LUCIA), Jurkat T-cells
(suspension cultures) were transduced with HIV-Luc recombinant
virus containing supernants at an MOI of 0.5 in the presence
of 8 ~zg/ml polybrene at 37°C for 1 hour. Cells were washed and
then plated in multiple wells (3 xlOQ cells/well) of a 96-well
opaque-white tissue culture plate (Corning) containing
different concentrations of inhibitors. For HeLa cells
(adherent cells) were plated and allowed to attach for 24
hours before exposure to virus containing supernatants. Cells
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were incubated at 37°C for 48 hours post virus addition and
Luciferase activity was quantified on a Packard TopCount-NXT
microplate scintillation counter using Bright-Glo luciferase
assay reagent (Promega Corp.). The standard error (S. E.) given
for all quantified transduction experiments is calculated from
at least three independent experiments. Cytotoxicity was
evaluated in parallel with LUCIA (but without virus) using the
commercially available CellTiter-96 AQ"e°US one solution cell
proliferation assay (Promega Corp.), according to the
manufacturers' instructions.
HIV-1 ~-day replication assays
C8166 T-cells were washed and infected with HIV-1 (strains
HXB2wt and HXB2AZTresr RT amino acid changes 67N, 70R, 215F,
219Q) at low multiplicity of infection for one hour at room
temperature. The cells were then washed and distributed (5 x
109 cells/well) in triplicate into the wells of 96-well cell
culture plates containing different concentrations of
inhibitors. The plates were then incubated at 37°C for 4 days.
The cell-free culture fluid was then harvested and assayed for
levels of p24 viral antigen using a commercially available
ELISA kit (Murex), according to the manufacturers'
instructions. Cytotoxicity was evaluated by distributing (5 x
109 cells/well) uninfected C8166 T-cells into triplicate wells
of 96-well cell culture plates containing different
concentrations of inhibitor and incubating the plates at 37°C.
After 4 days, 251 of XTT, which is metabolised by viable but
not dead cells was added and the plates incubated for a
further 3 hours at 37°C. Finally, the absorbance was read at a
wavelength of 450nm.
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Results
In vitro ATM assays
Compounds were assayed for ATM inhibition activity using the
method described above. Some results are detailed below in
Table 1 as ICSO values (the concentration at which 50% of the
enzyme activity is inhibited). These are determined over a
range of different concentrations, normally from 100 uM down
to 1 nM. Such ICso values are used as comparative values to
identify increased compound potencies.
TABLE 1
Compound ICSO ATM
-
4 <200 nM
5 <200 nM
6 <2 ~M
7 <200 nM
9 <20 uM
13 <20 uM
17 <200 nM
18 <200 nM
The following compounds had ICSO values of less than 200nM: 19-
43, 44-87, 93, 102, 106-107, 109-113, 115-117, 119, 122-124,
126-131, 133-135, 137-140, 142-182.
The following compounds had ICSO values of less than 2uM, in
addition to those listed above: 88-92, 94-101, 103-105, 108,
114, 118, 120-121, 125, 132, 136 and 141.
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Sensitisation of Cells to ionising radiation or DNA double
strand break chemotherapies
The data shown in figures 1-4 clearly show that inhibiting ATM
with compound 4 has a significant effect on sensitising tumour
derived cell lines to DNA double strand break inducing agents.
Retroviral transductions (LUCIA)
Compound 4 (known as Ku0064) was tested for its ability to
repress retroviral infections using HIV-1 based LUCIA (Fig.5).
It was found to efficiently inhibit HIV-1 LUCIA at low
micromolar concentrations in Jurkat T-cells (Fig. 5) as well
as all other ATM proficient cell lines tested. The 500
inhibitory concentration (ICSO) for Compound 4 in LUCIA was
around 1-2 uM in Jurkat cells (Fig. 5) and in the range of 1
to 10 uM for all other cell lines tested.
Compound 4 was also tested for cytotoxic and growth inhibition
effects in parallel to LUCIA to ensure that this was not the
reason for the observed reduction in transduction efficiency.
At concentrations up to 10 uM, compound 4 exposure showed no
significant cytotoxic effects on Jurkat cells during the
course of the assay (Fig. 5).
HIV-1 based LUCIA was performed on HeLa cells in the presence
of increasing concentrations of both compound 4 and the
nucleoside analog reverse transcriptase inhibitor, 3'-azido-
3'-deoxythymidine (AZT).
Fig. 7 shows that the combination of compound 4 and AZT was
found to act more effectively in inhibiting HIV-1 infection
than either drug alone. Fig. 7 provides an example in which
increasing concentrations of AZT was found to enhance the
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effectiveness of a compound of the present invention in
inhibiting HIV-1 infections.
HIV Replication Inhibition
A replication competent HIV-1 strain (HIVHxaz) was used to
infect C8166 T-cells in the absence or presence of increasing
concentrations of compound 9 (Figure 8), in order to
demonstrate the effectiveness of compounds of the present
invention in a system where HIV replication occurs. After 4-
days of virus replication, the amount of HIV-1 in cell culture
supernatants was quantified by p24 antigen ELISA. As a
control, the RT inhibitor AZT was used in parallel
experiments. Fig. 8A shows the inhibition of HIV-1
replication of a wild type HIV-1 strain (HIVHxsz wt) by compound
4 and AZT. The ICSO concentration of Compound 4 for HIV-1
replication inhibition was 0.1 uM. AZT showed an ICSO of 0.002
uM.
4-day replication assays were performed using an AZT drug
resistant strain of HIV-1 (HIVHxaz AZTres) in the absence or
presence of increasing concentrations of compound 4 (Fig. 8B,
Table 2). The ICSO concentration of Compound 4 for HIV-1
replication inhibition on the AZT resistant strain was 0.06
uM. AZT showed an ICso of 0.05 uM (Table 2), thereby
demonstrating a 25-fold resistance to AZT when compared to the
wild type strain.
Compound 4 inhibited HIV-1 replication equally well on wild-
type HIV-1 (ICSO = 0.1 uM; Fig. 8A, Table 2) as on the AZT
resistant HIV-1 strain (ICSO = 0.06 uM; Fig. 8B, Table 2).
These data provide evidence that compounds of the present
invention may be effective in both the treatment of wild-type
and acquired AZT resistant HIV-1 infections and, by
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implication, HIV-1 strains resistant to other drugs that
target viral proteins.
Compound 4 was also tested for cytotoxic and growth inhibition
effects on C8166 cells in parallel to the HIV-1 replication
assays to ensure that this was not the reason for the observed
effects of viral titre (Fig. 8C). Exposure of C8166 cells to
Compound 9 showed no significant cytotoxic effects during the
course of the assay or over the effective concentration range
(less than 1 uM) shown to inhibit HIV-1 replication. The 500
cytotoxic concentration (CCso) of compound 9 on C8166 cells was
estimated to be greater than 20 pM. Table 2 summarises the
experiments showing the anti-HIV-1 activity of Compound 4 in
4-day replication assays. Interestingly, the ICso in LUCIA (1
~M; Fig 5) was observed to be 10-fold higher than in the
replication assays (0.1 uM; Fig 8A). This difference can be
explained by the fact that in replication assays multiple
rounds of infection occur and each round provides the
potential for HIV-1 inhibition. Therefore, the inhibitory
effect of Compound 4 becomes compounded in HIV-1 replication
assays. The estimated ICso concentrations in replication
assays may therefore represent a more accurate reflection of
the extent of inhibition that may be seen in HIV-1 infected
patients.
Compound HIV-1 HXB2 ( ICSOuM) HIV-1 HXB2 ( ICSO~M)
Wild type AZT resistant
Compound 4 0.1 0.06
AZT 0.002 0.05
(25 fold resistant)
Table 2
